The Immunogenicity Study And Segmental Prokaryotic Expression Of ADV NS1Gene

Aleutian disease (AD) is a chronic, progressive, viral infectious disease caused by Aleutiandisease virus (ADV). There would induced acute interstitial pneumonia when young minks areinfected with AD; while adult minks are infected with AD, would result in decreased fertility ofmale minks, and miscarriage, stillbirth or mummified fetus of female minks. There have been noeffective vaccines or drugs to prevent and control the spread of AD, though periodic detectionand eliminate disease minks have been used to purify mink groups. The prevalence of AD hasbrought a huge economic losses to the mink farming households around the world. Therefore, itis important and significant to develop an effective vaccine.The ADV genome mainly consists of the non structural protein NS1and the structuralprotein VP2, the NS1protein plays a certain role in the virus replication and post-transcriptionalcontrol in vivo of mink, and the structural protein VP2is the major immunogenic protein of theADV. This experiment aims to research the nonstructural protein NS1. According to theGenbank login NS1sequence, we know that the NS1gene contains a section of80bp longintron sequence and the NS1protein can not be expressed continuously in prokaryoticexpression system, We used the gene splicing by overlap extension PCR (SOE-PCR) methodand designed overlapping complementary primers to make the NS1gene spliced into afull-length gene, which was carried on the analysis biological information. The hydrophilicity,surface accessibility, flexibility, protein secondary structure and antigenicity was predictedresearch by using DNAStar Protean module. The signal peptide and transmembrane region ofthe protein was predicted by Network resources; According to the results, the NS1gene wasdivided into five segments, and respectively cloned into pMD18-T or pGEM-T vectors; TheNS1full-length gene and five segment genes were respectively connected to the expressionvector pGEX-4T-1and recombinant expression plasmids were successfully constructed. It wasinduced expression by IPTG and SDS-PAGE results validate that all of the six genes weredetected on the sites of the expected size proteins; Western Blotting results have proved thateach protein has the reactogenicity. The six proteins were purified respectively to immunizemice, while PBS control groups and GST-tagged protein control groups were established to testmice humoral immune response and cellular immune response by indirect ELISA, flowcytometry assay, lymphocyte proliferation assay (CCK-8) and cytokine assay (IL-4and IFN-γ).The results showed that each of the groups had certain immunogenicity, NS1-2groups and NS1-3groups showed the stronger immunogenicity. This study has reference value for thesubsequent study on immune effect of mink, and lays the foundation for the development ofgene engineering subunit vaccine.