Establishment Of The Method To Distinguish The Anti-Avian Influenza Virus Antibody Between Vaccinated And Infected Chicken

Avian influenza (AI) is a highly contagious viral disease affecting therespiratory, digestive and nervous system of many species of birds. It iscaused by a Type A influenza virus of the orthomyxoviridae family. Thereare two types of avian influenza virus, low pathogenicity (LPAI) and highpathogenicity (HPAI) which is classified as one of A list disease by OfficeInternational des Epizooties (OIE). Since the first breakout in 1978, Highlypathogenic avian influenza A (HPAI) occurred in HongKong, Vietnam,Japan, south Korea and so on, brought serious economic loss to the poultryindustry, so it is important to establish a quick and accurate diagnosingmethod.A vaccine incorporating the inactivated whole-virus has been used inan attempt to prevent AI infection. While these traditional vaccines canprotect against clinical infection and death, they can interfere withsurveillance programs. Vaccination produces the same antibodies as anactual AI infection, and can commonly cause false positives withtraditional diagnostic tests. AIV has two kinds of Non-structural protein:NS1 usually exist in nucleus, but NS2 mainly exist in cytoplasm andsometimes in nucleus. The NS1 virus protein typically was found in largeamounts in virus-infected cells but not the virus itself. So if we monitoredboth infected and vaccinated poultry for antibodies reactive to NS1, whilelive virus infection induced high levels of the specific NS1 antibody,commercial vaccines (inactived vaccines) induced little or no antibodyresponse. In this study we try to establish an efficient method todifferentiate vaccinated poultry by commercial vaccine and wild type virusinfected poultry. In the research, we have extracted the total RNA of MDCK cell whichhad been infected by AIV through trizol-reagent-lysis method.Accordingto AIV A/Chicken/Korea/MS96 (H5N1)strain's nucleotide sequence,wedesigned a pair of specific primers, and through RT-PCR amplification, weobtained the NS1 gene target band, about 690bp, linked the recoveredtarget fragment with cloning vector pMD 18-T, transformed Ecoli.DH5αcompetent cell, picked white bacterial colony to extract plasmid,recombinant plasmid was sequenced after PCR characterizing, thehomology between target gene and AIV NS1 gene sequence in GENBANKwas above 95%, which showed we had obtained AIV H5N1 NS1 genethrough amplification. NS1 gene was subcloned into prokaryoutic high-expressing vectorpET-32a(+) with double enzyme digestion of BamHI/HindIII. We usedvector's general premier to sequence pET-32a(+)-NS1 recombinantplasmid. The result showed that the sequenced NS1 gene was identical tothe protosequence and the target gene which was inserted into expressingvector had not mutated, and had correct direction and site. BL21 competentcell was transformed by positive recombinant plasmid and was induced toexpress by IPTG.Expession product being analysized through SDS-PAGE,target protein and vector multimetric histidine protein were confluentlyexpressed, and a target band of 45.7kDa appeared. Then we studied theexpressing condition of recombinant protein. There are more recombinantproteins expressing with more time and 6h after being induced,recombinant protein reach its maximum. The expressing quantity ofrecombinant protein is influenced by the concentration of IPTG .In thecondition of 1mmol/L IPTG being induced 6 hours, the extrinsic protein ofbacteria is more than in any other conditions. After NS1 express somaticwas lysized through supersonics, we analysized supernatant and deposity.The result that most of expression product was in deposit showed thattarget protein was existed in cytorrhyctes form which has not activity. In order to obtain active protein of nature conformation, we optimizedthe condition for extraction and purification of recombinant protein fromcytorrhyctes of bacteria. Three methods to isolate and purificatecytorrhycte were designed. The first method was using urea solution tolysis cytorrhycte, afer that, using gradiently dialysis to dispose thesupernatant, so the recombinant protein would reach 50%-60%; Thesecond method was using the multimetric histidine lable of fusion protein,we would obtain high purity recombinant protein through Ni2+affinitychromatograpHy, after renaturing, protein purity can reach 80%-90%; Thethird method was dissolving cytorrhyctes by anionic detergent N-sodiumlauryl sarkosinate. Supernatant was dialysized in the solution containingbuffer A, we would obtain recombinant protein with 90%-98% purity.Comparing the three methods, we could conclude the third is the best. Thefusion protein of NS1 purified was analysized with SPF chicken's H5N1subunit AIV positive serum by Western Bloting, after that, NS1 expressionproduct appeared a clear band on 45.7kDa. The result showed that thismethod offered necessary experiment material and basis for further studyof HS1 strain NS1 viral constitutive protein's constitution and function. Itwill help to develop specific diagnosing kit. In our study, the fusion protein as envelope antigen acted with SPF...