Cloning And Differential Expression Analysis Of Hcher-1and HcMSP Genes In Haemonchus Contortus

Haemonchus contortus is a blood sucking nematode that infects sheep, goats and other ruminants worldwide. Infections with this parasite can cause anaemia, weight loss and death, especially in lambs. The current methods for the control of gastrointestinal nematodes rely heavily on chemicals, but this has led to an increase of anthelmintic-resistant parasites. Together with the growing concern over residual chemicals in the environment and food chain, the situation has resulted in the need to search for the development of alternative or supplementary means for control this parasite.Great progress has been made on the immunology of H. contortus. But we still have no commercial vaccines. Research on the biology such as the development and sex determination of H. contortus would be helpful for looking for alternative contol measues for this parasite. In this study, two male development related genes were cloned and parts of the characters were learned.1. Molecular cloning, expression and immunogenicity of Hcher-1from adult male H. contortusPartial of Hcher-1gene of H. contortus was cloned by RT-PCR, using a pair of gene pecific primers which based on the conserved sequence of C. elegans, C. briggsae and B. malayi. Then the5’and3’ends of Hcher-1gene were obtained by5’RACE and3’RACE method. After sequence analysis, the full sequence of Hcher-1gene was1091bp long, which contains ORF (960bp),5’UTR (67bp) and3’UTR (64bp). After which, the ORF was cloned and ligated to the expression plasmid pET-28a (+). The recombinant pET-28a (+)-Hcher-1was transford to E. coli DE3and induced by IPTG. The result of SDS-PAGE showed the recombinant protein (weigted about38kD) was expressed in the suspematant and inclusion body. By western blot, we found that the recombinant protein can be recognized by the serum from naturely infected goats. The recombinant protein was purified and administered into the rat. The anti-serum were used in the western bolt, the result showed that the natural Hcher-1protein of the adult H. contortus could be recognized.2. Molecular cloning, expression and characterization of HcMSP from adult male Haemonchus contortusPartial of HcMSP gene of H. contortus was cloned by RT-PCR, using a pair of gene pecific primers which based on the conserved sequence of C. elegans. Then the5’and3’ ends of HcMSP gene were obtained by5’RACE and3’RACE method. After sequence analysis, the full sequence of HcMSP gene was439bp long, which contains ORF (381bp),5’UTR (17bp) and3’UTR (41bp). After which, the ORF was cloned and ligated to the expression plasmid pET-28a (+). The recombinant pET-28a (+)-HcMSP was transford to E. coli DE3and induced by IPTG. The result of SDS-PAGE showed the recombinant protein (weigted about15kD) was expressed in the suspernatant and inclusion body. By western blot, we found that the recombinant protein can not be recognized by the serum from naturely infected goats. The recombinant protein was purified and administered into the rat. The anti-serum were used in the western bolt, the result showed that the natural HcMSP protein of the adult H. contortus could be recognized.3. Differential expression of Hcher-1and HcMSP in different life stages of H. contortusThree pairs of gene specific primers were designed based on the Hcher-1gene, HcMSP gene and housekeeping gene β-tubulin. Then the amplification efficiencies were tested. RNAs from egg, L3, adult male and female were extracted and quantitied by Real Time PCR. We took the2-△△CT method to detect the expression in different life stages. Results showed that Hcher-1expresses highest in the male, and HcMSP highest in the male. Hcher-1expresses lowest in the female, and HcMSP lowest in larve3and female.