对虾白斑综合症病毒囊膜蛋白基因vp26和vp28的克隆及在毕赤酵母中的表达
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摘要
对虾白斑综合症病毒(white spot syndrome virus,WSSV)是全球对虾的主要病原之一,对全球对虾养殖业造成了严重的损失。目前在分子水平WSSV的感染和致病机理还未阐明,结构和功能基因的分析以及致病机理研究是目前WSSV研究的热点。WSSV是一种具有囊膜的双链DNA病毒,VP26和VP28是它的两个主要的囊膜蛋白,其中VP28已被初步证明在病毒吸附与入侵中起着关键作用,VP26研究较少,但它和VP28是同源蛋白。本研究成功实现了vp26和vp28基因在毕赤酵母中的分泌表达,为以后制备特异性抗体,开展WSSV的快速有效检测和WSSV亚单位疫苗的研制奠定了基础,同时为探索外源基因的真核表达积累了经验。
1.vp26、vp28基因的克隆:根据GenBank库中对虾白斑病毒的全基因组序列,分别设计扩增该病毒主要结构蛋白基因vp26、vp28的相应引物,以从发病的斑节对虾(Penaeus monodon)中提取的总DNA为模板,利用PCR技术对目的基因进行了扩增,获得长度均为615bp的2个基因片段,片段大小与预期一致。将目的片段与pGEM-T-easy载体连接,导入大肠杆菌DH5α,获得阳性重组子pT-vp26、pT-vp28。目的片段测序结果与GenBank库中公布的vp26、vp28基因序列同源性达到了99%以上,说明已成功获得vp26和vp28基因片段,且基因在进化上非常保守。
2.重组蛋白在毕赤酵母中的分泌表达及表达产物的免疫活性:将vp26、vp28克隆至毕赤酵母穿梭表达质粒pPIC9K,构建重组表达载体pPIC9K-VP26、pPIC9K-VP28,经BglⅡ线性化酶切,电穿孔将其整合到毕赤酵母宿主菌GS115。重组毕赤酵母经PCR鉴定和G418筛选后进行甲醇诱导表达。表达产物分别进行SDS-PAGE、Western blot和ELISA分析,结果表明:所得到的vp26和vp28基因都能够进行分泌表达,表达产物大小均为31kDa左右;Western blot和ELISA结果表明鼠抗血清可与获得的重组蛋白发生特异性反应。
White spot syndrome virus (WSSV) is one of the main shrimp pathogens causing serious losses in shrimp aquaculture. At present, the infection and pathogenesis of WSSV on molecular level have not been illuminated. Research on structure and function genes and pathogenic mechanism of the virus is in hotspot. WSSV is an enveloped double-strand DNA virus, VP26 and VP28 are two major envelope proteins. VP28 had been proved to play a key role in the initial steps of WSSV invasion, and VP26 may perform the similar functions as VP28. In this study, recombinant proteins (VP26 and VP28) were successfully expressed and secreted from host Pichia Pastoris. The results will provide important foundation for future studies on preparation of specific antiserum, effective detection of WSSV and development of subunit vaccine. Besides, the results will help improving the methods for expression foreign genes in eukaryotic host.
1. Cloning of vp26 and vp28 genes: According to the complete genome sequences of White Spot Syndrome Virus (WSSV) published on GeneBank, two pairs of primers were designed for PCR amplification of genes vp26 and vp28. Both of the amplified products were 615bp. The amplified products were ligated into the pGEM-T-easy vectors and then transformed into competent cells of Escherichia coli DH5α. The positive recombinants were named pT-vp26 and pT-vp28 respectively. Sequencing of target fragments indicated that the sequences of vp26 and vp28 genes have more than 99% similarity with the published sequences in GenBank database, suggesting that the two genes are evolutionarily conserved.
2. Expression and immunoactivity of recombinant proteins in Pichia Pastoris: The vp26 and vp28 genes were inserted into Pichia Pastoris secretory expression vectors pPIC9K. The recombinant plasmids pPIC9K-VP26 and pPIC9K-VP28 were linearized with BglⅡ, and then were transformed into Pichia Pastoris GS115 via electroporation. After G418 selection and PCR confirmation, the transformants were induced with methanol (0.5%) at 30℃for target proteins expression. The expression products were subjected for analysis by SDS-PAGE, ELISA and Western blot. The results showed that the vp26/28 genes were successfully expressed and secreted from host Pichia Pastoris and the expression products were about 30kDa. The Western blot and the ELISA analysis indicated that the expressed protein could react specifically with mouse polyclonal anti-serum.
1.vp26、vp28基因的克隆:根据GenBank库中对虾白斑病毒的全基因组序列,分别设计扩增该病毒主要结构蛋白基因vp26、vp28的相应引物,以从发病的斑节对虾(Penaeus monodon)中提取的总DNA为模板,利用PCR技术对目的基因进行了扩增,获得长度均为615bp的2个基因片段,片段大小与预期一致。将目的片段与pGEM-T-easy载体连接,导入大肠杆菌DH5α,获得阳性重组子pT-vp26、pT-vp28。目的片段测序结果与GenBank库中公布的vp26、vp28基因序列同源性达到了99%以上,说明已成功获得vp26和vp28基因片段,且基因在进化上非常保守。
2.重组蛋白在毕赤酵母中的分泌表达及表达产物的免疫活性:将vp26、vp28克隆至毕赤酵母穿梭表达质粒pPIC9K,构建重组表达载体pPIC9K-VP26、pPIC9K-VP28,经BglⅡ线性化酶切,电穿孔将其整合到毕赤酵母宿主菌GS115。重组毕赤酵母经PCR鉴定和G418筛选后进行甲醇诱导表达。表达产物分别进行SDS-PAGE、Western blot和ELISA分析,结果表明:所得到的vp26和vp28基因都能够进行分泌表达,表达产物大小均为31kDa左右;Western blot和ELISA结果表明鼠抗血清可与获得的重组蛋白发生特异性反应。
White spot syndrome virus (WSSV) is one of the main shrimp pathogens causing serious losses in shrimp aquaculture. At present, the infection and pathogenesis of WSSV on molecular level have not been illuminated. Research on structure and function genes and pathogenic mechanism of the virus is in hotspot. WSSV is an enveloped double-strand DNA virus, VP26 and VP28 are two major envelope proteins. VP28 had been proved to play a key role in the initial steps of WSSV invasion, and VP26 may perform the similar functions as VP28. In this study, recombinant proteins (VP26 and VP28) were successfully expressed and secreted from host Pichia Pastoris. The results will provide important foundation for future studies on preparation of specific antiserum, effective detection of WSSV and development of subunit vaccine. Besides, the results will help improving the methods for expression foreign genes in eukaryotic host.
1. Cloning of vp26 and vp28 genes: According to the complete genome sequences of White Spot Syndrome Virus (WSSV) published on GeneBank, two pairs of primers were designed for PCR amplification of genes vp26 and vp28. Both of the amplified products were 615bp. The amplified products were ligated into the pGEM-T-easy vectors and then transformed into competent cells of Escherichia coli DH5α. The positive recombinants were named pT-vp26 and pT-vp28 respectively. Sequencing of target fragments indicated that the sequences of vp26 and vp28 genes have more than 99% similarity with the published sequences in GenBank database, suggesting that the two genes are evolutionarily conserved.
2. Expression and immunoactivity of recombinant proteins in Pichia Pastoris: The vp26 and vp28 genes were inserted into Pichia Pastoris secretory expression vectors pPIC9K. The recombinant plasmids pPIC9K-VP26 and pPIC9K-VP28 were linearized with BglⅡ, and then were transformed into Pichia Pastoris GS115 via electroporation. After G418 selection and PCR confirmation, the transformants were induced with methanol (0.5%) at 30℃for target proteins expression. The expression products were subjected for analysis by SDS-PAGE, ELISA and Western blot. The results showed that the vp26/28 genes were successfully expressed and secreted from host Pichia Pastoris and the expression products were about 30kDa. The Western blot and the ELISA analysis indicated that the expressed protein could react specifically with mouse polyclonal anti-serum.
引文
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