本文選題：犬瘟熱病毒 + H基因��； 參考：《江西農(nóng)業(yè)大學》2015年碩士論文

【摘要】：犬瘟熱病毒(canine distemper virus,CDV)是犬瘟熱(canine distemper,CD)的病原體。CD是威脅犬類健康的最嚴重傳染病之一,發(fā)病后死亡率極高,且無特異性治療藥物。為此,本試驗針對CDV的N基因,建立了快速診斷RT-PCR方法;同時通過RT-PCR方法擴增CDV部分H基因,通過原核表達、復性,成功表達出CDV H蛋白,并構(gòu)建了H蛋白間接ELISA方法,證實表達的H蛋白具有免疫原性,具體內(nèi)容如下:1.根據(jù)GenBank公布的CDV N蛋白基因序列,設(shè)計了一對特異性引物,成功擴增出464bp的CDV N基因片段。測序結(jié)果表明該序列與GenBank已經(jīng)發(fā)表CDV基因序列的同源性為94.8%~98.5%。利用本文建立的CDV檢測RT-PCR方法對30例疑似CDV病例檢測顯示:RT-PCR檢測方法的陽性檢出率為90%、膠體金試紙陽性檢出率為86.7%,說明RT-PCR方法檢出率和敏感性更優(yōu)。2.血凝素蛋白是誘導機體產(chǎn)生中和抗體的主要蛋白之一。本文去除H基因高度疏水區(qū)核苷酸設(shè)計引物,成功克隆了來自南昌CDV病料中CDV長度為1236nt的H基因片段。將該基因片段插入到載體pET32a中,構(gòu)建了原核表達質(zhì)粒pET32a-CDVH,重組蛋白H基因在Rosetta菌中獲得了高效表達。表達產(chǎn)物以包涵體的形式存在,經(jīng)包涵體洗滌、復性、Ni柱純化、透析濃縮,最終獲得濃度為0.561mg/m L重組H蛋白溶液。小鼠免疫試驗顯示,重組蛋白免疫產(chǎn)生的血清抗體效價在1:102400以上,免疫原性良好。3.以7.5ug/m L的復性H蛋白溶液包被96孔酶標板,成功建立了重組CDV H蛋白間接ELISA。間接ELISA條件為:重組蛋白包被濃度7.5ug/m L、1%明膠封閉、血清稀釋倍數(shù)為1:80、酶標二抗稀釋倍數(shù)為1:5000,陰陽性血清判定的臨界值(OD492)為0.100。試驗證明該間接ELISA方法特異性強,重復性良好。對收集的背景清楚血樣的檢測結(jié)果符合預期。
[Abstract]:Canine distemper virus (CDV) is the pathogen of canine distemper CD.CD is one of the most serious infectious diseases that threaten the health of dogs. Therefore, a rapid diagnostic RT-PCR method was established for the N gene of CDV, and the partial H gene of CDV was amplified by RT-PCR, and the CDV H protein was successfully expressed by prokaryotic expression and renaturation, and an indirect Elisa method of H protein was constructed. The expressed H protein has immunogenicity, as follows: 1. According to the sequence of CDVN protein gene published in GenBank, a pair of specific primers were designed to amplify the CDVN gene fragment of 464bp successfully. The sequencing results showed that the homology between this sequence and the published CDV gene sequence in GenBank was 94.898. 5%. Using the CDV detection RT-PCR method established in this paper, 30 suspected cases of CDV were detected by RT-PCR. The results showed that the positive detection rate of the two methods was 90 and 86.7 respectively, which indicated that the detection rate and sensitivity of RT-PCR was better than that of gold test paper. Hemagglutinin protein is one of the main proteins that induce the production of neutralizing antibodies. In this paper, the H gene fragment with length of 1236nt from Nanchang disease material was cloned successfully by removing the high hydrophobic nucleotide design primers of H gene. The gene fragment was inserted into the vector pET32a and the prokaryotic expression plasmid pET32a-CDVHwas constructed. The recombinant protein H gene was highly expressed in Rosetta bacteria. The expressed product existed in the form of inclusion body, washed by inclusion body, purified by renaturation Ni column, and concentrated by dialysis. Finally, the recombinant H protein solution with 0.561mg/m L concentration was obtained. The antibody titer of the recombinant protein was above 1: 102400, and the immunogenicity was good. 3. Indirect ELISAs of recombinant 7.5ug/m H protein were successfully established by coating 96 well enzyme labeled plates with H protein solution of 7.5ug/m L renaturation. The indirect Elisa conditions were as follows: the concentration of recombinant protein was blocked by 7.5ug/m 1% gelatin, the dilution multiple of serum was 1: 80, the dilution multiple of enzyme labeled second antibody was 1: 5 000, and the critical value of yin-yang serum was 0.100. The indirect Elisa method was proved to be specific and reproducible. The results of blood samples with clear background were in line with expectations.
【學位授予單位】：江西農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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