Complementary DNA Gene Cloning, Expression And Protective Immunity In Goats Of H11 Antigen From Haemonchus Contortus

Haemonchus contortus is an important gastrointestinal nematode that belongs to the Haemonchus genus of Trichostrongylidae family, which parasitizes ruminants such as goats, sheep, cattle and so on. The parasite feeds on host blood, causing severe anaemia which can be fatal to young animals, which lead significant economic loss of husbandry industry. Many helminthic were used for a long time and been proved for efficiently, but with the emergence and spread of anti-drug strains, classic methods for prevention of Haemonchus contortus was challenged.H11, the intergral membrance protein now widely known as a hidden antigen, from the intestinal microvilli of parasitic stages of Haemonchus contortus was identified in detergent extracts of brush borders. H11 preparations from adult worms were used to immune a number of different breeds of sheep in UK, Australia and South Africa. The dada from these experiments showed us that H11 was most effective immunogen isolated from Haemonchus contortus. The male, female worm burdens and faecal egg counts could be decreased by 70%, 80% and 90% respectively after immunization with this antigen.Complementary DNAs and PCR products coding for H11 have been cloned and sequenced. But in China few researching have been carried out in Haemonchus contortus H11, we commenced this scientific research as follows:1. Cloning, Identification and Sequences Analysis of H11 cNDA from Haemonchus contortusThree pairs of gene-specific primers were designed based on the sequences of H11 genes of Haemonchus contortus accepted in GenBank. Three products, amplified by reverse transcriptase- polymerase chain reaction (RT-PCR) with total RNA extractedfrom adult worms of Haemonchus contortus as template, were inserted to pMD18-T vector and transformed into is.coli JM109 respectively. The recombinant plasmids were identified by restriction enzymes digestion, and PCR. For completely identification of these genes, the nucleotide was sequenced and analyzed. 98% homology with H11 of Haemonchus contortus and 61% identity with Peptidase Ml family of Caenorhabditis elegans were found. The HEXXH and GAMEN motifs characteristic of microsomal aminopeptidase was also found when the deduced amino acid sequence was compared with that of other rhabditata and mammalians. The protein's second structure and antigenicities were analyzed and predicted at the same time.2. Expression and Activity Analysis of Recombinant H11 from Haemonchus contortusH11 gene of Haemonchus contortus were subcloned into pET-28(b) prokaryotic expression vector and the positive recombinant plasmids were obtained by restriction enzymes digestion and PCR amplification. Then these E. coli BL21 carrying plasmid pET-H11-1 , pET-H11-2 and pET-H11-3 were induced by IPTG SDS-PAGE gel showed that 39kD H11-1 and 42kD H11-2 were expressed and the recombinant proteins were formed as inclusion bodies, but pET-H11-3 did not expressed. The recombinant proteins in inclusion bodies were denaturalized by 8mol/L urea and then dialyzed in PBS containing degressive concentration of urea step by step, after which, these refolding proteins were purified by affinity chromatography. Aminopeptidase activities of the recombinant protein was found and it could be inhibited by phenanthroline but not by phenylmethylsulfonyl floride or ethylenediaminetetraceate.3. Immunity of Recombinant Haemonchus contortus H11 in GoatsTwenty 4-6 months old lambs, of similar weight, were allocated to 5 groups. For lambs in group 5 was controlled by given no challenge or immunization. The other 16 lambs in group 1, 2, 3 and 4 were immunized twice with PBS, recombinant H11-1, H11-2 and H11-l+H11-2 about 28 and 14 days before challenge by 10000 Haemonchus contortus L3, respectively. Serums from the goats were tested by ELISA, it was found that the antibodies against H11 reach a high level at 14 days after primary immunization, while arrived at the peak value at 10 days post boosting, and then keep a high level for a long time. In this experiment, the levels of cytokins such as interleukin 2, interleuk...