| In recent years, due to the duck virus hepatitis （DVH） transmission is fast, strongpathogenic, serotype variation, seriously restrict the development of the duck industry.Atpresent, the detection of the disease is the most commonly used method of polymerase chainreaction （PCR） and enzyme-linked immunosorbent assay （ELISA）.But these methods thereare operation tedious, time consuming, low sensitivity, poor accuracy. Therefore to establish akind of can be applied to duck viral hepatitis clinical samples rapid, simple and standardizedtesting technology is imminent.PCR-ELISA methods had been suggested at the turn of the century. PCR and ELISA werecombined in the method, and introduce specific probe, in effectively simplifies the operationsteps of qualitative and quantitative analysis of the samples at the same time, greatly improvingthe sensitivity and specificity of detection results. PCR and ELISA techniques in virusclassification, pathogenic bacteria detection, identification and molecular biology researchcloning has its unique advantages, applied to the detection of duck viral hepatitis, has certainresearch significance and promotion value.Around the world all have different degree of duck viral hepatitis, at present there is nocertain cure for this disease also. With the continuous variation of the DHV virus and DHV therapid development of molecular biology and genetic engineering technology, from the aspectssuch as safety, high efficiency, the promotion of genetic engineering vaccine will have goodprospects.IL-2is a well known immune therapy, immune enhancement, the immune preventionof cytokines, now we have a large number of experimental reports, IL-2used in the treatment ofanimal diseases to report for duty. This study cloned from duck hepatitis b virus genotype VP1gene, established the detection DHV PCR ELISA method, and through the complementarywould build the expression vector pET-VP1-IL-2, the prokaryotic expression, in order to furtherstudy the duck viral hepatitis diagnosis and prevention and control method laid a foundation.The results of the study are as follows:This test successfully cloned duck hepatitis b virus genotype VP1gene, into e. coli DH5alpha, the sequencing analysis found that the sequence size of517bp. Comparison ofhomologous sequence analysis found that the test of duck hepatitis virus VP1gene fragmentsand published in GenBank duck hepatitis b virus genotype VP1（EF502171.1EF502172.1）nucleotide sequence homology of87.56%. According to the position relations in the phylogenetic tree: amplification of duck hepatitis virus VP1nucleotides and published inGenBank database of duck hepatitis b virus genotype VP1gene sequence in the same branch,speculated that belong to the same species. But with a database of published nucleotidesequence of genotype Ⅲ belong to different phylogenetic tree branches, kinship relations faraway.This test probe primers double marking method is used to successfully established theduck viral hepatitis PCR ELISA detection method. And10mg/mL was determined by therepeated optimization chain avidin package is liquid,1%BSA sealing fluid,0.5pmol/L probeand1:4000dilution enzyme standard resistance and optimal hybrid optimum reactionconditions, such as time. This method is compared with conventional rt-pcr sensitivity up to100~1000times, and specificity is strong, for the duck viral hepatitis epidemiological studiesand clinical diagnosis provides a new way.The success of the experiment by a pair of would construct prokaryotic expression vectorof pET-VP1-IL-2, strain, and transformation to the expression by IPTG induction, sds-pageelectrophoresis analysis, obtained the purpose of VP1protein, the protein is mainly exists in theform of inclusion body, is about40.6KD molecular size. When the concentration of IPTG end of0.9mmol/L,4h,37℃VP1protein expression of the highest. |
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