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  本文選題：豬傳染性胃腸炎病毒 + 桿狀病毒載體表達系統(tǒng)�。� 參考：《南陽師范學院》2016年碩士論文

【摘要】：豬傳染性胃腸炎(Porcine transmissible gastroenteritis, TGE)是由豬傳染性胃腸炎病毒(Porcine transmissible gastroenteritis virus, TGEV)引起的豬的一種以腹瀉、嘔吐、脫水等為主要癥狀的高度接觸性消化道傳染病,對兩周齡以內的仔豬致死率可達100%,給全球養(yǎng)豬業(yè)造成了巨大危害。在TGEV基因組中,ORF2編碼的S蛋白攜帶有主要的B細胞抗原識別決定簇,經研究證實S蛋白N端的543個氨基酸殘基內含有A、B、C、D四個抗原位點,是唯一能誘導機體產生中和抗體的主要結構蛋白。因此,S蛋白的表達對TGEV基因工程疫苗和疾病診斷試劑的研制具有重要意義。本研究根據(jù)已發(fā)表的TGEVS基因cDNA序列,設計并合成了3對特異性引物,通過PCR擴增得到4個含有不同抗原位點的S基因片段：含D位點的S2基因,含A位點的S3基因,含A和D位點的S(2+3)基因以及含有A、B、C和D位點的Sfull基因。將S基因分別克隆到pMD19-Tsimple載體并進行測序鑒定。用DNA　star軟件將測序結果中不同S基因與Genebank中收錄的TGEV　S基因參考序列相比較,其核苷酸同源性為100%。將測序驗證正確的S基因的不同片段分別克隆到桿狀病毒轉移載體pFBDMY-IM中,隨后將獲得的陽性重組轉移載體通過Tn7轉座重組至桿狀病毒Bacmid中,然后將重組Bacmid轉染Sf9昆蟲細胞獲得重組病毒。將重組病毒感染Sf9昆蟲細胞,72小時后用熒光顯微鏡進行觀察,發(fā)現(xiàn)Sf9細胞出現(xiàn)明顯的細胞病理變化和紅色熒光,說明重組桿狀病毒已經成功構建。提取重組桿狀病毒基因組DNA并用TGEV S基因的特異性引物進行PCR鑒定,結果證實TGEV S基因已經成功重組至桿狀病毒基因組中。用SDS-PAGE和Western Blot分析蛋白表達產物,結果表明：S2、S3、Sfull基因在桿狀病毒表達系統(tǒng)中成功得到表達,表達的重組蛋白大小分別為25.9 KDa、27.5KDa和85.1KDa；S(2+3)基因在桿狀病毒表達系統(tǒng)中沒有表達。因此,本研究將含不同抗原位點的S基因片段在桿狀病毒表達系統(tǒng)中進行表達,為以后利用S蛋白開發(fā)TGEV基因工程疫苗和疾病診斷試劑奠定了基礎。
[Abstract]:Porcine transmissible enteritisis (TGEV) is a highly contagious infectious disease of the digestive tract caused by porcine transmissible gastroenteritis virus (TGEV), which is characterized by diarrhea, vomiting, dehydration, etc. The fatality rate of piglets within two weeks of age can reach 100, which has caused great harm to the global pig industry. In the TGEV genome, the S protein encoded by ORF2 carries the main B cell antigen recognition determinant. The 543 amino acid residues at the N-terminal of S protein contain four antigenic sites of Agna Bu CfU D. It is the only major structural protein that can induce the production of neutralizing antibodies. Therefore, the expression of S protein plays an important role in the development of TGEV gene engineering vaccine and disease diagnosis reagent. According to the published cDNA sequence of TGEVS gene, three pairs of specific primers were designed and synthesized. Four S gene fragments containing different antigenic sites were amplified by PCR: S2 gene containing D locus and S3 gene containing A locus. The Szc23) gene with A and D loci and the Sfull gene with Agna C and D loci. S gene was cloned into pMD19-Tsimple vector and sequenced. DNA star software was used to compare the different S genes in the sequencing results with the reference sequences of TGEV S gene included in Genebank. The nucleotide homology was 100%. The different fragments of the correct S gene were cloned into the baculovirus transfer vector pFBDMY-IM, and the obtained positive recombinant transfer vector was recombined into the baculovirus Bacmid by Tn7 transposition. Then the recombinant Bacmid was transfected into Sf9 insect cells to obtain the recombinant virus. After 72 hours of infection with recombinant virus, Sf9 insect cells were observed by fluorescence microscope. It was found that there were obvious cellular pathological changes and red fluorescence in Sf9 cells, which indicated that the recombinant baculovirus had been successfully constructed. The genomic DNA of recombinant baculovirus was extracted and identified by PCR with specific primers of TGEV S gene. The results showed that the TGEV S gene had been successfully recombined into the genome of baculovirus. SDS-PAGE and Western Blot were used to analyze the protein expression products. The results showed that the cell S2S3Sfull gene was successfully expressed in the baculovirus expression system, and the expressed recombinant protein was 25.9 KDa-27.5KDa and 85.1 KDA-Sf23) gene, which was not expressed in the baculovirus expression system. Therefore, the S gene fragments with different antigenic sites were expressed in baculovirus expression system, which laid a foundation for the development of TGEV gene engineering vaccine and disease diagnosis reagent by using S protein.
【學位授予單位】：南陽師范學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S852.651

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