本文選題：口蹄疫病毒 + 豬細(xì)小病毒��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：口蹄疫對我國養(yǎng)殖業(yè)造成嚴(yán)重威脅,由于傳統(tǒng)滅活疫苗存在病毒毒力返強(qiáng)、滅活不徹底等不安全因素,促使人們研究更加安全的口蹄疫疫苗。PPV-VLPs亞單位疫苗具有類似天然病毒的穩(wěn)定性和免疫原性,不具有病毒核酸,沒有感染性,因此具有很好的應(yīng)用前景。本研究以P1代重組病毒為材料,研究重組病毒增值工藝,利用桿狀病毒表達(dá)系統(tǒng),研究重組蛋白最適表達(dá)條件及免疫原性,以期研發(fā)出廉價(jià)、高效、安全的PPV-VLPs亞單位疫苗。本文通過對比昆蟲細(xì)胞懸浮培養(yǎng)和貼壁培養(yǎng)2種方式下的細(xì)胞形態(tài),研究昆蟲細(xì)胞的最佳培養(yǎng)方式;通過用血球計(jì)數(shù)板每隔24h進(jìn)行細(xì)胞計(jì)數(shù),繪制懸浮細(xì)胞復(fù)制動力學(xué)曲線,研究昆蟲昆蟲細(xì)胞生長周期。為了得到高滴度的P3代重組病毒,將P1代重組病毒轉(zhuǎn)染昆蟲細(xì)胞,PCR鑒定插入的外源基因是否丟失;Western-blotting檢測重組蛋白表達(dá)及其反應(yīng)原性;采用不同的MOI感染昆蟲細(xì)胞,不同時(shí)間收獲病毒,通過測定病毒度,研究重組病毒增值工藝。采用不同的MOI感染昆蟲細(xì)胞,不同感染時(shí)間收獲細(xì)胞沉淀,進(jìn)行Western-blotting分析,用軟件對比灰度值,研究重組蛋白最適表達(dá)條件。采用最佳重組蛋白表達(dá)條件感染大量的懸浮昆蟲細(xì)胞,進(jìn)行重組蛋白的大量表達(dá),將粗純的蛋白表達(dá)產(chǎn)物加佐劑乳化肌肉注射豚鼠,采集血清,用間接ELISA方法檢測抗體水平。結(jié)果表明,(1)昆蟲細(xì)胞的最佳培養(yǎng)方式是懸浮培養(yǎng);(2)為了得到高滴度的P3代重組病毒,PCR鑒定插入的外源基因沒有因?yàn)閭鞔鴣G失;(3)研究重組病毒增值工藝,得出最佳MOI是0.05個(gè)MOI,最佳感染時(shí)間是72h;(4)重組蛋白在昆蟲細(xì)胞中獲得較好的表達(dá),Western-blotting分析結(jié)果表明重組蛋白的反應(yīng)原性良好,同時(shí)進(jìn)一步證明了插入的口蹄疫病毒中和表位的存在;(5)研究重組蛋白最適表達(dá)條件,Westernblotting分析結(jié)果表明最適條件是10個(gè)MOI,最佳感染時(shí)間72h;(6)間接ELISA方法檢測結(jié)果表明,滅活疫苗組和空衣殼免疫組都產(chǎn)生了較低水平的FMDV中和抗體和較高水平的PPV特異性抗體。(7)用SPSS軟件對間接ELISA結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析,進(jìn)一步驗(yàn)證了PPV空衣殼免疫原性。
[Abstract]:Foot-and-mouth disease (FMD) poses a serious threat to the breeding industry in China. Because of the unsafe factors such as the virus virulence and inactivation of traditional inactivated vaccines, It is suggested that a more safe foot-and-mouth disease vaccine. PPV-VLPs subunit vaccine has the similar stability and immunogenicity of natural viruses, no viral nucleic acid and no infection, so it has a good prospect of application. In this study, P1 generation recombinant virus was used as the material, the recombinant virus value-added technology was studied, and the optimal expression conditions and immunogenicity of recombinant protein were studied by using baculovirus expression system, in order to develop a cheap, efficient and safe PPV-VLPs subunit vaccine. In this paper, by comparing the morphology of insect cell suspension culture with that of adherent culture, we studied the optimum culture method of insect cell, and plotted the dynamic curve of suspension cell replication by counting cells every 24 hours with blood cell counter. The growth cycle of insect cells was studied. In order to obtain the high titer P3 generation recombinant virus, the P1 generation recombinant virus was transfected into insect cells to identify whether the inserted foreign gene was missing or not, and to detect the expression of recombinant protein and its reactivity by Western-blotting. The virus was harvested at different time, and the technology of recombinant virus increment was studied by measuring the virus degree. The optimal expression conditions of recombinant protein were studied by using different MOI infected insect cells and harvested cells at different time of infection. Western-blotting analysis was carried out and the gray value of recombinant protein was compared with software. A large number of suspended insect cells were infected with the best recombinant protein expression condition. The crude protein expression product was injected into guinea pig muscle with adjuvant emulsification. The serum was collected and the antibody level was detected by indirect ELISA method. The results showed that the best culture method for insect cells was suspension culture. In order to obtain high titer P3 recombinant virus, the inserted foreign gene was not lost due to passage. The results of Western-blotting analysis showed that the optimal MOI was 0. 05 moi, and the best infection time was 72 hau 4). The results of Western-blotting analysis showed that the reactivity of the recombinant protein was good. Furthermore, the existence of neutralizing epitopes of the inserted foot-and-mouth disease virus (FMDV) was confirmed.) the optimal expression conditions of recombinant proteins were analyzed by Western blotting. The results of Western blotting showed that the optimal conditions were 10 moi and the best time of infection was 72hmHX6) indirect ELISA method was used to detect the recombinant protein. Both the inactivated vaccine group and the empty capsid immunization group produced lower levels of FMDV neutralizing antibody and a higher level of PPV specific antibody. The results of indirect ELISA were statistically analyzed by SPSS software, which further verified the immunogenicity of PPV empty capsid.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 李偉;秦紅剛;張萍;漆世華;朱薇;王威;謝紅玲;溫文生;吳玉石;;Sf9昆蟲細(xì)胞懸浮培養(yǎng)工藝研究[J];中國獸藥雜志;2012年08期

2 羅莉;李坤;王保成;王明蓉;;包涵體變復(fù)性技術(shù)研究進(jìn)展[J];中國醫(yī)藥生物技術(shù);2012年04期

3 于少芳;吳小鋒;;提高昆蟲桿狀病毒表達(dá)系統(tǒng)重組蛋白表達(dá)水平的技術(shù)[J];蠶桑通報(bào);2010年03期

4 甄洪花;沈志強(qiáng);單虎;王金良;付石軍;張松林;;豬細(xì)小病毒結(jié)構(gòu)蛋白VP2研究進(jìn)展[J];動物醫(yī)學(xué)進(jìn)展;2009年08期

5 張佑紅;陳龍;靖志強(qiáng);鄭偉;陳林;秦琴;;不同周期Sf9細(xì)胞琥珀酸脫氫酶酶活的研究[J];武漢工程大學(xué)學(xué)報(bào);2009年05期

6 崔保安;魏戰(zhàn)勇;王學(xué)斌;黃克和;金喜新;董振杰;鄭蘭蘭;;IL-2與豬細(xì)小病毒VP_2基因雙表達(dá)載體的構(gòu)建及免疫原性的研究[J];生物工程學(xué)報(bào);2006年03期

7 李文建,沈明山,李春江,陳亮;口蹄疫抗原決定簇融合基因轉(zhuǎn)化胡蘿卜的研究[J];廈門大學(xué)學(xué)報(bào)(自然科學(xué)版);2005年S1期

8 易詠竹,陳寅,張志芳,何家祿,秦儉;宿主域擴(kuò)大的重組救活昆蟲桿狀病毒表達(dá)載體的構(gòu)建及外源基因的表達(dá)[J];蠶業(yè)科學(xué);2002年04期

9 王曉遲,戚藝華,歐陽藩;Sf9細(xì)胞的培養(yǎng)工藝[J];化工冶金;1998年03期

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