Construction And Analysis Of Subtractive CDNA Libraries Between Differential Immunogenicity Strains Of Eimeria Maxima

Avian coccidiosis is a parasitic protozoal disease caused by several species of Eimeria coccidian which parasitize in intestinal mucosa cells. Coccidiosis has the highest incidence in avian diseases, and it is also one of the most important disease of domestic fowl. Current control of coccidiosis is based on the use of chemotherapeutic agents. However, the prolonged drug use has led to the emergence of drug resistance strains in the field, and the efficiency of drugs is becoming lower and lower. The long-term use of coccidiostats not only lead to be residual in eggs and to pollute environment, but also endanger human health. So the controlling of coccidiosis is going to from drugs to immune prevention. Eimeria maxima is one of the most common coccidians in field which has the best immunogenicity and well cross protection. So it is the indispensable component in coccidial vaccine. But E.maxima has the most significant antigenic variation in different strains. Sometimes, because of antigentic diversity between vaccine strain and field strain, the immune efficiency will be low and even failed. The researches of our group in former time found that E.maxima SH strain and E.maxima NT strain had significant differences in immunogenicity. SH strain produced immune protection only to homologous strain, and NT strain produced immune protection both to homologous and heterogeneous strain. In order to study the molecule basis of the differences in immunogenicity between these two strains, the researchs were carried out as follows.1Construction of subtractive cDNA libraries of E. maxima Shanghai and Nantong Strains in unsprolated stageThe total RNA of E. maxima Shanghai(SH) and Nantong(NT) strains were extracted from unsproluted oocysts using routine method. The mRNA was purified with cellulose column, and then it was used to reverse transcript to cDNA. The cDNA from SH strain was used as tester and the cDNA from NT strain was used as driver, respectively, the cDNA from NT strain was used as tester and the cDNA from SH strain was used as driver when suppression subtractive hybridization(SSH) was used to constructe two subtractive cDNA libraries. After two cycles of subtractive hybridization and two cycles of suppressed PCR, the amplificated products were ligated to pGEM-T Easy vector, and then conversed to competent Escherichia coli cells. The positive clones were identificated with blue/white selection. To confirm that the cloned cDNA fragments are indeed present in Escherichia coli cells, PCR with nest primer1and nest primer2R. which were designed respectively from Adaptor1and2R. were employed to detect561clones. The results showed that the recombination rate of the inserted fragment was95%(284/300)in SH strain, and was89%(231/261) in NT strain. The length of inserted fragments in both libraries ranged from0.25kb to1.0kb.2Selection and analysis of differentially expressed genes in subtractive cDNA libraries of E. maxima SH and NT strains in unsprolated stageFirstly,4kinds of DIG-labeled probes namely subtractive and unsubtractive DIG-Labeled cDNA probes of SH strain, subtractive and unsubtractive DIG-Labeled cDNA probes of NT strain were prepared. Sencondly, the same clone was detected by both subtractive DIG-Labeled cDNA probe which came from homologous strain and unsubtractive DIG-Labeled cDNA probe which came from heterogenetic strain. A total of86differentially expressed clones which contained63clones of SH strain and23clones of NT strain were obtained from515positive clones which contained284clones of SH strain and231clones of NT strain. According to nucleotide sequence analysis of these86clones,10contigs were obtained, and6contigs came from SH strain and4contigs came from NT strain, respectively. Among the10contigs,5contigs in6contigs of SH strain and3in4contigs of NT strain shared significant identity with previously described or hypothetical proteins, respectively, and1contigs in both liabraries had not found identity proteins, which were presumed to be new sequences. Finally,6differentially expressed genes were detected by reverse transcription PCR, and were showed that these genes expressed in both strains. Real Time PCR was employed to quantify the relative abundance of2in6differentially expressed genes, and were showed that these2genes had differently expression in SH strain and NT strain.