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Construction of Subtractive cDNA Libraries of the Sporogony Stage of Eimeria tenella by Suppression Subtractive Hybridization
CHINESE JOURNAL OF BIOTECHNOLOGY Volume 23, Issue 6, November 2007 Online English edition of the Chinese language journal Cite this article as: Chin J Biotech, 2007, 23(6), 1005í1010.
RESEARCH PAPER
Construction of Subtractive cDNA Libraries of the Sporogony Stage of Eimeria tenella by Suppression Subtractive Hybridization HAN Hong-Yu, LIN Jiao-Jiao, ZHAO Qi-Ping, DONG Hui, JIANG Lian-Lian, WANG Xin, HAN Jing-Fang, HUANG Bing* Key Laboratory for Animal Parasitology of Ministry of Agriculture / Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200232,China
Abstract:
To clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from
unsporulated oocysts and sporulated oocysts of E. tenella were used as the driver, respectively, the cDNAs from sporozoites of E. tenella was used the tester. Two subtractive cDNA libraries of sporozoites were constructed using the suppression subtractive hybridization (SSH) technique. The cDNAs from unsporulated oocysts was used the driver, the cDNAs from sporulated oocysts was used the tester and one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximately 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively. Thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with the former. A total of 40 unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with the former and the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis. Key Words: Eimeria tenella, unsporulated oocysts, sporulated oocysts, sporozoite, suppression subtractive hybridization
Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflicts severe economic losses on the poultry industry at an estimated worldwide annual loss of more than US$3 billion[1]. At present, conventional disease control strategies have mainly relied on prophylactic medication. However, a novel control approach is hoped to substitute prophylactic medication, due to the emergence of drug-resistant parasites and safety of food. The live oocyst vaccines are useful alternatives to prophylactic medication, but represent some disadvantages, such as the difficulty of conservation, the diffusion of species, the danger of potential
coccidiosis, which were not widely used on poultry. Novel control approaches are urgently needed to study to control coccidiosis effectively. The life cycles of Eimeria are complex, and it is likely that gene expression profiles in Eimeria differ significantly between differentially developmental stages. Until now, some genes of differential developmental stages had been cloned and isolated, and the function of these genes had been studied. Some genes only expressed on one developmental stage of the life cycles; for example, invasion-related protein[2,3]. These differentially expressed genes may be related to invasion,
Received: February 26, 2007; Accepted: May 16, 2007. * Corresponding author. Tel: +86-21-54113251; E-mail: [email protected] This work was supported by the grants from the National Natural Technology Resource Sharing Platform (No. 2005DKA21205-4) and the National High Technology Research and Development Program (863 Program, No. 2006AA10A207-1). Copyright © 2007, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
growth, developmental and proliferation of Eimeria. Isolation and identification of differentially expressed genes from the different developmental stages of Eimeria systematically may be provided important rationale to find new targets for studying new vaccines and drugs against coccidiosis. Eimeria tenella is one of the most important species causing avian coccidiosis, and is used as a mode species to study Eimeria spp. To analyze differentially expressed genes of E.tenella sporozony stages overall, two subtractive cDNA libraries of sporozoites and one library of sporulated oocysts were constructed using the technique of suppression subtractive hybridization (SSH). Here, 50 clones were selected to sequence from three libraries, respectively. ESTs were analyzed in Genbank with Blast search.
1
Materials and methods
1.1 Parasites E. tenella was originally provided by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Parasites were maintained and propagated as previously described[4]. E. tenella was maintained by passage through coccidia-free two-week-old chickens. Unsporulated oocysts were obtained from the caecal contents of chickens eight days post infection. Some unsporulated oocysts were purified and then stored in liquid nitrogen, and some were incubated in 2.5% potassium dichromate solution. After sporulation, the sporulated oocysts were collected and purified. One half of the sporulated oocysts were stored in liquid nitrogen. The remaining was used to purify sporozoites according the reported[5]. 1.2 Isolation of total RNA and ploy(A)+ RNA Total RNA were extracted from unsporulated oocysts, sporulated oocysts and sporozites, respectively, using TRizol (Invitrogen). Ploy(A)+RNAs were isolated using Oligotex® mRNA Mini Kit (Qiagen). All procedures were done according to the manual provided. Total RNA and ploy(A)+ RNA were each quantified by UV-spectrophotometry (Eppendorf), the integrity of total RNA and ploy(A)+ RNA was assessed on a denaturing , formaldehyde 1% agarose/EtBr gel . 1.3 Suppression subtractive hybridization The SSH procedure was performed with a PCR-selectTM cDNA subtraction kit (Clontech) as directed by the manufacturer, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as the driver, respectively, and the cDNAs from sporozoites of E. tenella was used the tester. Two subtractive cDNA libraries of sporozoites were constructed using the technique of suppression subtractive hybridization (SSH). The cDNAs from unsporulated oocysts was used as the driver, the cDNAs from sporulated oocysts was used as the tester, and one subtractive cDNA library of sporulated oocysts was
constructed. 1.4 Constructed and Preserved of three subtractive cDNA libraries After two hybridizations of the double-stranded cDNAs, three pools of subtracted cDNAs were amplified by nested PCR according to the manufacturer’s recommended protocol. The PCR products were cut from the gel and purified using the QIAquick®Gel Extraction kit (Qiagen). Purified PCR products were ligated into pGEM-T-easy vector (Promega) overnight at 4°C with T4 DNA ligase. Ligations were transformed into JM109 and plated on LB with ampicillin, X-gal, and IPTG. Selected white colonies were grown overnight in LB with ampicillin and stored in 96-well plates. Fifty white clones were randomly amplified with SP6 and T7 primers from three subtractive cDNA libraries, respectively to determine the recombinant efficiency and the size of fragments. 1.5 Sequencing and analysis Fifty clones were randomly selected and sequenced from three subtractive cDNA libraries of the sporogony stages of E.tenella, respectively. These ESTs were analyzed in GenBank with blast search.
2
Results
2.1 Isolation and analysis of RNA The integrity of RNA was examined by the electrophoresis samples. Total RNA of three stages of E. tenella exhibits two bright bands corresponding to ribosomal 28S and 18S RNA, respectively (Fig. 1), indicating good quality. The ratios of OD260/OD280 of total RNA were 1.9, suggesting little contamination of polysaccharides and proteins. These results indicated that RNA could be used to isolate ploy(A)+and synthesize first-stand cDNA.
Fig. 1 Isolation of total RNA from sporogony stage of E. tenella 1: total RNA of sporozoites; 2: total RNA of unsporulated oocysts; 3: total RNA of sporulated oocysts.
2.2 Double-stand cDNAs synthesis and Rsa I digestion Double-stranded cDNAs were synthesized independently
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
with Reverse Transcriptase and cDNA synthesis primer from the tester and driver, respectively, and further digested with a four-base-cutting restriction enzyme Rsa I that yielded blunt ends. The tester cDNAs were then subdivided into two portions (1 and 2) and each were ligated to a different double-stranded adapter (adapters 1or 2R), respectively. After digestion with Rsa I, double-stranded cDNAs were checked using agarose/EtBr gel electrophoresis, which indicated that the size of fragments was between 0.1 kb to 2.0 kb (Fig. 2).
primers. These results indicated that two subtractive cDNA libraries of sporozoites and one library of sporulated oocysts contained approximately 96%, 96%, and 98% recombinant clones, respectively, the sizes of fragments were between 500 bp to 1 kb (Fig. 3). 2.4 Sequence and analysis Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively. Thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described (Table 1) and these genes include mucin-associated surface protein [Trypanosoma cruzi], acidic microneme protein [E. tenella], putative PDI-like protein [Toxoplasma gondii] and so on. A total of 40 unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described (Table 2), including protein serine/threonine phosphatases [Polaromonas sp.], calciumdependent protein kinase 2 (PfCDPK2) [Plasmodium falciparum], cleavage and polyadenylation specificity factor, subunit [Theileria annulata] and so on. The other sequences represent novel genes of E. tenella with no significant homology to the proteins in GenBank.
3
Discussion
Fig. 2 Double strands cDNA and double strands cDNA after Rsa I digestion 1: ds cDNA of sporulated oocysts; 2: ds cDNA of sporulated oocysts after Rsa I digestion; 3: ds cDNA of unsporulated oocysts; 4: ds cDNA of unsporulated oocysts after Rsa I digestion; 6: ds cDNA of sporozoites; 7: ds cDNA of sporozoites after Rsa I digestion; 5: DNA size markers (DL2000, from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp).
2.3 Construction and identification of subtractive cDNA libraries After the final panning procedure, the binding specificity of phage-displayed peptides with rotavirus particles was judged by ELISA. One hundred and ninety clones were randomly picked for ELISA procedure. Twenty-nine positive clones were selected, amplified, and further identified by ELISA. Of the 29 phage clones, 10 showed highly positive results indicting their strong binding with rotavirus with BSA as control (Fig. 1). The results indicated that peptide Nos. 2 and 8 occupied the maximum absorbance values (>0.890) of all peptides, but all the values were larger than 0.660, a strong signal indicating their binds. The nested PCR products were ligated into pGEM-T-easy vector (Promega), and then transformed into JM109. Three subtractive libraries of sporogony stage of E. tenella were constructed; two libraries of sporozoites and one of sporulated oocysts. Fifty clones were randomly selected to PCR amplify with SP6 and T7
Suppression Subtractive Hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. It is based primarily on a suppression PCR technique and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared[6]. Compared to other methods used to select differentially expressed genes, it has many good qualities, such as high specificity, high sensitivity, high efficiency, widely used to select differentially expressed genes, concentrated mainly on the medical science, but seldom on the parasitology. At present, it was used to study genes on the important parasites including Plasmodium[7], Leishmania[8], Schistosoma[9], Eimeria[10] and so on. The life cycles of parasites are very complex, including some development stages with different morphological characteristic and living environment. So each stage has differentially expressed genes. Until now, some differentially expressed genes of different development stages of harmful parasites were found by SSH. For example, to select differentially expressed genes of the oocyst sporozoites and salivary gland sporozoites in Anopheles mosquitoes, highly up-regulated mRNAs transcribed in salivary gland sporozoites were identified using suppression subtractive cDNA hybridization[11].
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
Fig. 3 PCR identification of recombinant plasmids from three subtractive cDNA libraries M: marker DL2000 (DL2000, from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); 1–16: the two subtractive cDNA libraries of sporozoites; 17–24: the subtractive cDNA library of sporulated oocysts. Table 1 Comparison of some clones in subtractive cDNA libraries of sporulated oocysts by sequence homologization in GenBank Clone
Best match in database
Clone number
BW1-A01
Mucin-associated surface protein (MASP) [Trypanosoma cruzi]
2
BW1-A06
E. tenella mRNA for acidic microneme protein
17
BW1-A09
Microneme protein precursor Etmic-1 [E. tenella]
7
BW1-B02
Small heat shock protein 21 [Toxoplasma gondii]
1
BW1-B05
TA4 antigen protein [E. tenella]
8
BW1-E03
E. tenella mRNA for surface antigen 13
4
BW1-E06
Putative PDI-like protein [Toxoplasma gondii]
1
BW1-G08
Hypothetical protein, expressed [Toxoplasma gondii RH]
1
Table 2 Comparison of some clones in two subtractive cDNA libraries of sporozoites by sequence homologization in GenBank Clone
Best match in database
Clone number
ZB1-A08
Protein serine/threonine phosphatases [Polaromonas sp.]
8
ZB1-H07
Calcium-dependent protein kinase 2 (PfCDPK2)[P. falciparum]
2
ZB1-H08
Hypothetical protein PFL0830w [P. falciparum 3D7]
1
ZB10-A04
Cleavage and polyadenylation specificty factor, subunit [Theileria annulata strain Ankara]
1
ZB10-B04
Hypothetical protein e2017b09.tmp0045 [E. tenella str. Houghton]
1
ZB10-E05
sphingomyelin/lysocholinephospholipid -phospholipase C-related [P. yoelii ]
1
ZW10-A06
Cystathionine beta-synthase (Serine sulfhydrase) (Beta-thionase )
1
ZW1-B07
SERPIN1 protein precursor [E. tenella]
3
ZW10-D09
ABC transporter, substrate-binding protein [Bordetella avium 197N]
1
Selection of differentially expressed genes of parasites using SSH would help to study the relationship between structure and function, and find genes related to growth and development. Using SSH to select differentially expressed genes of parasites after medication help to study the mechanism of anti-drugs. Maybe, novel targets could be found for prevention and cure of parasitic diseases. To ensure the effection of the SSH, it is very important to select samples with identical and similar genetic background and living environment. In this study, three development stages of E. tenella, unsporolated oocysts, sporulated oocysts
and sporozoites, were used to identify differentially expressed genes. The quality of RNA is very crucial for successful experiment. Total RNA cannot be used to synthesize cDNA, 1–2 mg of poly(A)+RNA is required per subtraction. During experiment, attention must be paid to hybridization times. First, hybridization time was 6–12 h, if the time was too long, almost all single-strand cDNAs were combined with each other, and so new hybrid molecules were not formed which comprise of differentially expressed cDNAs with different adaptors on each end. If the time is too short, single-strand
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
cDNAs of the tester were not normalized enough, so some low-abundance differentially expressed cDNAs could be lost. The second hybridization time was 12–24 h; to ensure low-abundance differentially expressed genes could be formed double-stand cDNAs hybrids. In this study, the results indicate that 10 h for the first hybridization and 20 h for the second were reasonable. The most abundant transcripts isolated from sporulated oocysts encodes the microneme proteins which are secreted from apicomplexan parasites during invasion of host cells and play crucial roles in parasite-host cell adhesion[12]. Etmic-2 is one of the most abundant transcripts, encoding 50 kD acidic protein. The protein was found within the microneme organelles of E. tenella sporozoites and merozoites, and was only expressed on the surface of parasites during both sporozony and schizogony stages. During invasion host cell, the protein was focused at the point of parasite entry and secreted from the host-parasite interface. An hour after sporozoites enter the host cells, Etmic-2 protein was not detected on the host cell surface[13]. Rayn et al[14] reported microneme protein expression of E. tenlla, 12 h after sporulation and lasted 24 h, but was not detected in unsporulated oocysts. These results indicate that microneme proteins are very critical for parasite to invade host cells. Homologous protein serine/threonine phosphatases were one of the most abundant transcripts from two subtractive cDNA libraries of sporozoites. Serine/threonine phosphatases, an apozymase regulating reversible phosphorylation of protein, plays an important role with protein kinases in control of protein phosphorylation controlling a large variety of cellular events, such as cell metabolism, DNA replication, genetic transcription, RNA splicing, signal transduction, cell differentiation and apoptosis, oncogene transformation and so on. Previous studies had shown that the development of Plasmodium in cell was regulated with protein phosphorylation of host and parasite. Li et al[15] isolated a gene encoding Serine/threonine phosphatases of Plasmodium falciparum [PfPPJ]. It was the only expression in the sexual erythrocytic stage (gametocytes). After, Dobson et al[16] found that the PfPPJ was expressed on the surface of schizont, ring and trophozoite of P.falciparum, in the following pattern: schizont > ring > trophozoite. These results indicated that PfPPJ may be involved in sexual stage development and suggested expression of the PfPPJ was regulated by parasitic stage-specific transcription. At present, there are no reports about serine/threonine phosphatases of E. tenella. But Eimeria spp. is one of the apicomplexan parasites with Plasmodium. Perhaps, serine/threonine phosphatases of Eimeria may be expressed in some development stages of life cycles, and are involved in invasion host cells and development. These results confirmed that suppression subtractive hybridization can be used to select differentially expressed genes of the development stages of parasites. In
future, the study of these differentially expressed genes would help to understand the mechanism of invasion , growth and development of E.tenella. REFERENCES [1]
Shirley MW, Ivens A, Gruber A, et al. The Eimeria genome projects: a sequence of events. Trends Parasitol, 2004, 20(5): 199–201.
[2]
Peroval M, Pery P, Labbe M. The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth. Int J Parasitol., 2006, 36(10-11): 1205–1215.
[3]
Wiersma HI, Galuska SE, Tomley FM, et al. A role for coccidian cGMP-dependent protein kinase in motility and invasion. Int J Parasitol, 2004, 34(3):369–380.
[4]
Jiang JL, Jiang JS. An improved method for purification Eimeria tenella oocysts, sporozoites and second generation merozoites. Journal of China Agriculture University, 1996, 5(1): 99-102.
[5]
Song CP, Shen YL, Wang ZK. Cultivation of Eimeria tenella in vitro. Chinese Journal of Veterinary Science, 1998, 18(2): 48–52.
[6]
Diatchenko L, Lau YF, Campbell AP, et al. Suppression subtractive
hybridization:
A
method
for
generating
differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025–6030. [7]
Dessens JT, Margos G, Rodriguez MC, et al. Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization. Parasitology Today, 2000, 16(8): 354–356.
[8]
Bellatin JA, Murray AS, Zhao M, et al. Leishmania mexicana: identification of genes that are preferentially expressed in amastigotes. Exp Parasitol., 2002, 100(1): 44–53.
[9]
Nowak TS, Woodards AC, Jung Y, et al. Identification of transcripts
generated
during
the
response
of
resistant
Biomphalaria glabrata to Schistosoma mansoni infection using suppression subtractive hybridization. J Parasitol, 2004, 90(5): 1034–1040. [10] Han HY, Zhao QP, Jiang LL, et al. Construction of subtractive cDNA libraries of Eimeria tenella to Maduramycin and Diclazuril by suppression subtractive hybridization. Scientia Agricultura Sinica, 2005, 38(8): 1712–1716. [11] Matuschewski K, Ross J, Brown SM, et al. Infectivityassociated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J Biol Chem, 2002, 277(44): 41948–41953. [12] Tomley FM, Soldati DS. Mix and match module: structure and function of microneme proteins in apicomplexan parasites. Trends in Parasitology, 2001, 17: 81–88. [13] Tomley FM, Bumstead JM, Billington KJ, et al. Molecular cloning and characterization of a novel acidic microneme protein (Etmic-2) from the apicomplexan protozoan parasite, Eimeria tenella. Mol Biochem Parasito., 1996, 79(2): 195–206.
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
[14] Ryan R., Shirley M, Tomley FM. Mapping and expression of
Plasmodium falciparum. Eur J Biochem, 1997, 249(1): 98–106.
microneme genes in Eimeria tenella. Int J Parasitol, 2000, 30:
[16] Dobson S, Bracchi V, Chakrabarti D, et al. Characterization of
1493–1499. [15] Li JL, Baker DA. Protein phosphatase beta, a putative type-2A protein phosphatase from the human malaria parasite
a novel serine/threonine protein phosphatase (PfPPJ) from the malaria parasite, Plasmodium falciparum. Mol Biochem Parasitol , 2001, 115(1): 29–39.
RESEARCH PAPER
Construction of Subtractive cDNA Libraries of the Sporogony Stage of Eimeria tenella by Suppression Subtractive Hybridization HAN Hong-Yu, LIN Jiao-Jiao, ZHAO Qi-Ping, DONG Hui, JIANG Lian-Lian, WANG Xin, HAN Jing-Fang, HUANG Bing* Key Laboratory for Animal Parasitology of Ministry of Agriculture / Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200232,China
Abstract:
To clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from
unsporulated oocysts and sporulated oocysts of E. tenella were used as the driver, respectively, the cDNAs from sporozoites of E. tenella was used the tester. Two subtractive cDNA libraries of sporozoites were constructed using the suppression subtractive hybridization (SSH) technique. The cDNAs from unsporulated oocysts was used the driver, the cDNAs from sporulated oocysts was used the tester and one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximately 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively. Thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with the former. A total of 40 unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with the former and the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis. Key Words: Eimeria tenella, unsporulated oocysts, sporulated oocysts, sporozoite, suppression subtractive hybridization
Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflicts severe economic losses on the poultry industry at an estimated worldwide annual loss of more than US$3 billion[1]. At present, conventional disease control strategies have mainly relied on prophylactic medication. However, a novel control approach is hoped to substitute prophylactic medication, due to the emergence of drug-resistant parasites and safety of food. The live oocyst vaccines are useful alternatives to prophylactic medication, but represent some disadvantages, such as the difficulty of conservation, the diffusion of species, the danger of potential
coccidiosis, which were not widely used on poultry. Novel control approaches are urgently needed to study to control coccidiosis effectively. The life cycles of Eimeria are complex, and it is likely that gene expression profiles in Eimeria differ significantly between differentially developmental stages. Until now, some genes of differential developmental stages had been cloned and isolated, and the function of these genes had been studied. Some genes only expressed on one developmental stage of the life cycles; for example, invasion-related protein[2,3]. These differentially expressed genes may be related to invasion,
Received: February 26, 2007; Accepted: May 16, 2007. * Corresponding author. Tel: +86-21-54113251; E-mail: [email protected] This work was supported by the grants from the National Natural Technology Resource Sharing Platform (No. 2005DKA21205-4) and the National High Technology Research and Development Program (863 Program, No. 2006AA10A207-1). Copyright © 2007, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
growth, developmental and proliferation of Eimeria. Isolation and identification of differentially expressed genes from the different developmental stages of Eimeria systematically may be provided important rationale to find new targets for studying new vaccines and drugs against coccidiosis. Eimeria tenella is one of the most important species causing avian coccidiosis, and is used as a mode species to study Eimeria spp. To analyze differentially expressed genes of E.tenella sporozony stages overall, two subtractive cDNA libraries of sporozoites and one library of sporulated oocysts were constructed using the technique of suppression subtractive hybridization (SSH). Here, 50 clones were selected to sequence from three libraries, respectively. ESTs were analyzed in Genbank with Blast search.
1
Materials and methods
1.1 Parasites E. tenella was originally provided by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Parasites were maintained and propagated as previously described[4]. E. tenella was maintained by passage through coccidia-free two-week-old chickens. Unsporulated oocysts were obtained from the caecal contents of chickens eight days post infection. Some unsporulated oocysts were purified and then stored in liquid nitrogen, and some were incubated in 2.5% potassium dichromate solution. After sporulation, the sporulated oocysts were collected and purified. One half of the sporulated oocysts were stored in liquid nitrogen. The remaining was used to purify sporozoites according the reported[5]. 1.2 Isolation of total RNA and ploy(A)+ RNA Total RNA were extracted from unsporulated oocysts, sporulated oocysts and sporozites, respectively, using TRizol (Invitrogen). Ploy(A)+RNAs were isolated using Oligotex® mRNA Mini Kit (Qiagen). All procedures were done according to the manual provided. Total RNA and ploy(A)+ RNA were each quantified by UV-spectrophotometry (Eppendorf), the integrity of total RNA and ploy(A)+ RNA was assessed on a denaturing , formaldehyde 1% agarose/EtBr gel . 1.3 Suppression subtractive hybridization The SSH procedure was performed with a PCR-selectTM cDNA subtraction kit (Clontech) as directed by the manufacturer, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as the driver, respectively, and the cDNAs from sporozoites of E. tenella was used the tester. Two subtractive cDNA libraries of sporozoites were constructed using the technique of suppression subtractive hybridization (SSH). The cDNAs from unsporulated oocysts was used as the driver, the cDNAs from sporulated oocysts was used as the tester, and one subtractive cDNA library of sporulated oocysts was
constructed. 1.4 Constructed and Preserved of three subtractive cDNA libraries After two hybridizations of the double-stranded cDNAs, three pools of subtracted cDNAs were amplified by nested PCR according to the manufacturer’s recommended protocol. The PCR products were cut from the gel and purified using the QIAquick®Gel Extraction kit (Qiagen). Purified PCR products were ligated into pGEM-T-easy vector (Promega) overnight at 4°C with T4 DNA ligase. Ligations were transformed into JM109 and plated on LB with ampicillin, X-gal, and IPTG. Selected white colonies were grown overnight in LB with ampicillin and stored in 96-well plates. Fifty white clones were randomly amplified with SP6 and T7 primers from three subtractive cDNA libraries, respectively to determine the recombinant efficiency and the size of fragments. 1.5 Sequencing and analysis Fifty clones were randomly selected and sequenced from three subtractive cDNA libraries of the sporogony stages of E.tenella, respectively. These ESTs were analyzed in GenBank with blast search.
2
Results
2.1 Isolation and analysis of RNA The integrity of RNA was examined by the electrophoresis samples. Total RNA of three stages of E. tenella exhibits two bright bands corresponding to ribosomal 28S and 18S RNA, respectively (Fig. 1), indicating good quality. The ratios of OD260/OD280 of total RNA were 1.9, suggesting little contamination of polysaccharides and proteins. These results indicated that RNA could be used to isolate ploy(A)+and synthesize first-stand cDNA.
Fig. 1 Isolation of total RNA from sporogony stage of E. tenella 1: total RNA of sporozoites; 2: total RNA of unsporulated oocysts; 3: total RNA of sporulated oocysts.
2.2 Double-stand cDNAs synthesis and Rsa I digestion Double-stranded cDNAs were synthesized independently
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
with Reverse Transcriptase and cDNA synthesis primer from the tester and driver, respectively, and further digested with a four-base-cutting restriction enzyme Rsa I that yielded blunt ends. The tester cDNAs were then subdivided into two portions (1 and 2) and each were ligated to a different double-stranded adapter (adapters 1or 2R), respectively. After digestion with Rsa I, double-stranded cDNAs were checked using agarose/EtBr gel electrophoresis, which indicated that the size of fragments was between 0.1 kb to 2.0 kb (Fig. 2).
primers. These results indicated that two subtractive cDNA libraries of sporozoites and one library of sporulated oocysts contained approximately 96%, 96%, and 98% recombinant clones, respectively, the sizes of fragments were between 500 bp to 1 kb (Fig. 3). 2.4 Sequence and analysis Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively. Thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described (Table 1) and these genes include mucin-associated surface protein [Trypanosoma cruzi], acidic microneme protein [E. tenella], putative PDI-like protein [Toxoplasma gondii] and so on. A total of 40 unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described (Table 2), including protein serine/threonine phosphatases [Polaromonas sp.], calciumdependent protein kinase 2 (PfCDPK2) [Plasmodium falciparum], cleavage and polyadenylation specificity factor, subunit [Theileria annulata] and so on. The other sequences represent novel genes of E. tenella with no significant homology to the proteins in GenBank.
3
Discussion
Fig. 2 Double strands cDNA and double strands cDNA after Rsa I digestion 1: ds cDNA of sporulated oocysts; 2: ds cDNA of sporulated oocysts after Rsa I digestion; 3: ds cDNA of unsporulated oocysts; 4: ds cDNA of unsporulated oocysts after Rsa I digestion; 6: ds cDNA of sporozoites; 7: ds cDNA of sporozoites after Rsa I digestion; 5: DNA size markers (DL2000, from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp).
2.3 Construction and identification of subtractive cDNA libraries After the final panning procedure, the binding specificity of phage-displayed peptides with rotavirus particles was judged by ELISA. One hundred and ninety clones were randomly picked for ELISA procedure. Twenty-nine positive clones were selected, amplified, and further identified by ELISA. Of the 29 phage clones, 10 showed highly positive results indicting their strong binding with rotavirus with BSA as control (Fig. 1). The results indicated that peptide Nos. 2 and 8 occupied the maximum absorbance values (>0.890) of all peptides, but all the values were larger than 0.660, a strong signal indicating their binds. The nested PCR products were ligated into pGEM-T-easy vector (Promega), and then transformed into JM109. Three subtractive libraries of sporogony stage of E. tenella were constructed; two libraries of sporozoites and one of sporulated oocysts. Fifty clones were randomly selected to PCR amplify with SP6 and T7
Suppression Subtractive Hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. It is based primarily on a suppression PCR technique and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared[6]. Compared to other methods used to select differentially expressed genes, it has many good qualities, such as high specificity, high sensitivity, high efficiency, widely used to select differentially expressed genes, concentrated mainly on the medical science, but seldom on the parasitology. At present, it was used to study genes on the important parasites including Plasmodium[7], Leishmania[8], Schistosoma[9], Eimeria[10] and so on. The life cycles of parasites are very complex, including some development stages with different morphological characteristic and living environment. So each stage has differentially expressed genes. Until now, some differentially expressed genes of different development stages of harmful parasites were found by SSH. For example, to select differentially expressed genes of the oocyst sporozoites and salivary gland sporozoites in Anopheles mosquitoes, highly up-regulated mRNAs transcribed in salivary gland sporozoites were identified using suppression subtractive cDNA hybridization[11].
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
Fig. 3 PCR identification of recombinant plasmids from three subtractive cDNA libraries M: marker DL2000 (DL2000, from up to down: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); 1–16: the two subtractive cDNA libraries of sporozoites; 17–24: the subtractive cDNA library of sporulated oocysts. Table 1 Comparison of some clones in subtractive cDNA libraries of sporulated oocysts by sequence homologization in GenBank Clone
Best match in database
Clone number
BW1-A01
Mucin-associated surface protein (MASP) [Trypanosoma cruzi]
2
BW1-A06
E. tenella mRNA for acidic microneme protein
17
BW1-A09
Microneme protein precursor Etmic-1 [E. tenella]
7
BW1-B02
Small heat shock protein 21 [Toxoplasma gondii]
1
BW1-B05
TA4 antigen protein [E. tenella]
8
BW1-E03
E. tenella mRNA for surface antigen 13
4
BW1-E06
Putative PDI-like protein [Toxoplasma gondii]
1
BW1-G08
Hypothetical protein, expressed [Toxoplasma gondii RH]
1
Table 2 Comparison of some clones in two subtractive cDNA libraries of sporozoites by sequence homologization in GenBank Clone
Best match in database
Clone number
ZB1-A08
Protein serine/threonine phosphatases [Polaromonas sp.]
8
ZB1-H07
Calcium-dependent protein kinase 2 (PfCDPK2)[P. falciparum]
2
ZB1-H08
Hypothetical protein PFL0830w [P. falciparum 3D7]
1
ZB10-A04
Cleavage and polyadenylation specificty factor, subunit [Theileria annulata strain Ankara]
1
ZB10-B04
Hypothetical protein e2017b09.tmp0045 [E. tenella str. Houghton]
1
ZB10-E05
sphingomyelin/lysocholinephospholipid -phospholipase C-related [P. yoelii ]
1
ZW10-A06
Cystathionine beta-synthase (Serine sulfhydrase) (Beta-thionase )
1
ZW1-B07
SERPIN1 protein precursor [E. tenella]
3
ZW10-D09
ABC transporter, substrate-binding protein [Bordetella avium 197N]
1
Selection of differentially expressed genes of parasites using SSH would help to study the relationship between structure and function, and find genes related to growth and development. Using SSH to select differentially expressed genes of parasites after medication help to study the mechanism of anti-drugs. Maybe, novel targets could be found for prevention and cure of parasitic diseases. To ensure the effection of the SSH, it is very important to select samples with identical and similar genetic background and living environment. In this study, three development stages of E. tenella, unsporolated oocysts, sporulated oocysts
and sporozoites, were used to identify differentially expressed genes. The quality of RNA is very crucial for successful experiment. Total RNA cannot be used to synthesize cDNA, 1–2 mg of poly(A)+RNA is required per subtraction. During experiment, attention must be paid to hybridization times. First, hybridization time was 6–12 h, if the time was too long, almost all single-strand cDNAs were combined with each other, and so new hybrid molecules were not formed which comprise of differentially expressed cDNAs with different adaptors on each end. If the time is too short, single-strand
HAN Hong-Yu et al. / Chinese Journal of Biotechnology, 2007, 23(6): 1005–1010
cDNAs of the tester were not normalized enough, so some low-abundance differentially expressed cDNAs could be lost. The second hybridization time was 12–24 h; to ensure low-abundance differentially expressed genes could be formed double-stand cDNAs hybrids. In this study, the results indicate that 10 h for the first hybridization and 20 h for the second were reasonable. The most abundant transcripts isolated from sporulated oocysts encodes the microneme proteins which are secreted from apicomplexan parasites during invasion of host cells and play crucial roles in parasite-host cell adhesion[12]. Etmic-2 is one of the most abundant transcripts, encoding 50 kD acidic protein. The protein was found within the microneme organelles of E. tenella sporozoites and merozoites, and was only expressed on the surface of parasites during both sporozony and schizogony stages. During invasion host cell, the protein was focused at the point of parasite entry and secreted from the host-parasite interface. An hour after sporozoites enter the host cells, Etmic-2 protein was not detected on the host cell surface[13]. Rayn et al[14] reported microneme protein expression of E. tenlla, 12 h after sporulation and lasted 24 h, but was not detected in unsporulated oocysts. These results indicate that microneme proteins are very critical for parasite to invade host cells. Homologous protein serine/threonine phosphatases were one of the most abundant transcripts from two subtractive cDNA libraries of sporozoites. Serine/threonine phosphatases, an apozymase regulating reversible phosphorylation of protein, plays an important role with protein kinases in control of protein phosphorylation controlling a large variety of cellular events, such as cell metabolism, DNA replication, genetic transcription, RNA splicing, signal transduction, cell differentiation and apoptosis, oncogene transformation and so on. Previous studies had shown that the development of Plasmodium in cell was regulated with protein phosphorylation of host and parasite. Li et al[15] isolated a gene encoding Serine/threonine phosphatases of Plasmodium falciparum [PfPPJ]. It was the only expression in the sexual erythrocytic stage (gametocytes). After, Dobson et al[16] found that the PfPPJ was expressed on the surface of schizont, ring and trophozoite of P.falciparum, in the following pattern: schizont > ring > trophozoite. These results indicated that PfPPJ may be involved in sexual stage development and suggested expression of the PfPPJ was regulated by parasitic stage-specific transcription. At present, there are no reports about serine/threonine phosphatases of E. tenella. But Eimeria spp. is one of the apicomplexan parasites with Plasmodium. Perhaps, serine/threonine phosphatases of Eimeria may be expressed in some development stages of life cycles, and are involved in invasion host cells and development. These results confirmed that suppression subtractive hybridization can be used to select differentially expressed genes of the development stages of parasites. In
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