| Hypodermosis is a kind of serious zoonosis, which can produce huge economical loss to animal husbandry every year.The correlative studies indicate that Hypoderomin A (HA), Hypodermin B (HB) and Hypodermin C (HC) are the candidate antigens of serodiagnose and vaccine of Hypodermosis. The first instar larvae beening harvested was difficult, so it was fewer and fewer for natural protein used to the study of serodiagnose and immunity prevention of Hypodermosis. In order to obtain more purified recombinant protein, the study realized the expression in E.coli and purification of the three kinds of immune correlative protein, and developed the ELISA for Hypodermosis. All these will make a good foundation for the latter researches.Total RNA was extracted from the first instar larvae of H.lineatum, and then the first strand of cDNA was synthesized by reverse transcription. The specific primers of HA, HB and HC genes were designed according to the gene cDNA sequences reported in Genebank. The specific fragments were amplified by PCR and ligated into pMD19-T simple vector, then sequenced. Recombinant expression vector (pETHA, pETHB, pETHC) were constructed through subcloning the interested genes (HA, HB, HC) into the prokaryotic expression vector pET-30a(+) and transformed into BL21(DE3). The recombinant E.coli. was induced by IPTG in optimal culture condition, and then the expressed products was tested by SDS-PAGE and Western blotting. Inclusion body was extracted from those three kinds of induced recombinant E.coli.. The purified recombinant protein was obtained by denaturalization in 8M urea, dialysis or SEC renaturalization and Sephadex G-75 purification. The ELISA for Hypodermosis was developed using natural protein and recombinant protein as antigen. Conclusions can be drawed as follows:1. Total RNA was extracted from the first instar larvae of H.lineatum, and the 697bp of HA, 697bp of HB and 715bp of HC genes cDNA were amplified by RT-PCR.2. Recombinant cloning vectors of pTHA, pTHB and pTHC were constructed by ligating the HA, HB and HC genes cDNA into pMD19-T simple vector.3. In comparison with Genbank data, the homologies of the nucleotide sequences and amino acid sequences were the following: HA was 100% (L24914), 99.71% (X74303) and 100% (L24914), 99.56% (X74303); HB was 98.97% (L24915), 98.97% (X74304), 95.74% (X74305) and 98.23% (L24915), 98.23% (X74304), 93.36% (X74305); HC was 100% (AB054066), 99.42% (X74306) and 100% (AB054066), 99.57% (X74306), respectively. So there were high homologies between larvae strains in this study and those reported overseas.4. Recombinant prokaryotic expression vectors (pETHA, pETHB, pETHC) were constructed, and then HA, HB and HC genes were expressed successfully in E.coli. The three kinds of recombinant protein were expressed with the form of inclusion body and their expression amount reached 35%, 30% and 30% of the total protein, respectively.5. Western blotting showed: the rHA, rHB and rHC could specially combine with positive cattle serum IgG, so expression products have immunocompetence.6. The rHA, rHB and rHC were renaturalized by dialysis at 0.2mg/ml,0.5mg/ml and 1.0mg/ml concentration respectively. The three kinds of purified protein were obtained by Sephadex G-75 column.7. The rHA, rHB and rHC could be renaturalized by SEC.8 The ELISA for Hypodermosis was developed using natural protein and rHC protein as antigen. The specificity was 100% and 95.8%, respectively.The sensitivity were 98.36% and 100%, respectively.9. 233 sera samples collected from Inner Mongolia Region were examined by the ELISA for Hypodermosis using natural protein and rHC. The infected rate were 22.3%(52/233)and 20.6% (48/233), respectively. The positive coincidence rate was 88.46% (46/52) and 93.75% (45/48), respectively.10 The ELISA for Hypodermosis with rHC has higher respectivity and sensitivity, and it was better than that with natural protein on sensitivity and the positive coincidence rate. The rHC solved the shortage of natural antigen, so it is the optimal surrogate antigen of natural antigen.
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