摘要
为了在Sf9昆虫细胞中表达猪圆环病毒2型(PCV-2)Rep蛋白,试验以PCV-2毒株(PCV-2b)的DNA为模板扩增ORF1基因,将ORF1基因插入到转移载体质粒pQB3s上,构建重组质粒pQB3s-PCV-2b-Rep,将重组质粒与杆状病毒表达载体qBac-ⅢG共同转染Sf9昆虫细胞,获得P0代重组杆状病毒,经连续传代获得P1、P2、P3代重组杆状病毒,将P3代重组杆状病毒接种到Sf9昆虫细胞中进行悬浮培养,收集细胞并利用镍柱纯化重组表达的蛋白,通过荧光显微镜观察感染重组杆状病毒的Sf9昆虫细胞中的荧光量,通过SDS-PAGE分析和Western-blot鉴定重组蛋白的表达情况。结果表明:经PCR鉴定在945 bp处出现特异性条带;通过荧光显微镜观察,在转染后第1天就出现绿色荧光,第4~5天出现大量绿色荧光,P2、P3代重组杆状病毒感染Sf9昆虫细胞及P3代重组杆状病毒感染悬浮培养的Sf9昆虫细胞后在第4~5天出现大量绿色荧光;P2、P3代重组杆状病毒感染的Sf9昆虫细胞的细胞裂解液及纯化的重组Rep蛋白,通过SDS-PAGE分析和Western-blot鉴定在约38 ku处出现目的条带。说明试验成功构建出重组质粒pQB3s-PCV-2b-Rep及重组杆状病毒,构建的重组杆状病毒可以在悬浮培养的Sf9昆虫细胞中表达重组蛋白Rep。
In order to express Porcine circovirus type 2(PCV-2) Rep protein in Sf9 insect cells, the DNA of PCV-2 strain(PCV-2b) was used as template to amplify the whole ORF1 gene. The whole ORF1 gene was inserted into the transfer vector plasmid(pQB3s) to construct the recombinant plasmid pQB3s-PCV-2b-Rep. The recombinant plasmid and baculovirus expression vector(qBac-ⅢG) were co-transfected into Sf9 insect cells to obtain the P0 generation recombinant baculovirus. The recombinant baculoviruses of P1, P2 and P3 generation were obtained by continuous passage. The recombinant baculoviruses of P3 generation were inoculated into Sf9 insect cells for suspension culture. The cells were collected and the recombinant proteins were purified by Ni column. Fluorescence in Sf9 insect cells infected with recombinant baculovirus was observed by fluorescence microscopy. The expression of recombinant protein were analyzed by SDS-PAGE and Western-blot. The results showed that the specific bands at 945 bp were detected by PCR. Fluorescence microscopy showed that green fluorescence appeared on day 1 after transfection and reached the maximum on day 4 to 5. Sf9 insect cells infected with baculovirus of successive generations P1,P2 and P3 and Sf9 insect cells in suspension culture infected with P3 generation necombinant baculovirus showed a large amount of green fluorescence on day 4. The target band at 38 ku was identified by SDS-PAGE analysis and Western-blot identification in the cell lysate supernatant and purified recombinant protein Rep of Sf9 insect cells infected with P2 and P3 recombinant baculoviruses. These results indicated that the recombinant plasmid PQB3s-PCV-2b-Rep and the recombinant baculovirus were successfully constructed, and the recombinant baculovirus could expressed recombinant protein Rep in SF9 insect cells in suspension culture.
作者
梁太润
熊谋康
王爽云
张歆明
王志朋
王磊
刘献辉
于林洋
刘燕玲
张乐宜
宋长绪
LIANG Tairun;XIONG Moukang;WANG Shuangyun;ZHANG Xinming;WANG Zhipeng;WANG Lei;LIU Xianhui;YU Linyang;LIU Yanling;ZHANG Leyi;SONG Changxu(College of Animal Science,National Engineering and Technology Research Center for Pig Seed Industry,South China Agricultural University,Guangzhou 510642,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2023年第1期7-13,122,123,共9页
Heilongjiang Animal Science And veterinary Medicine
基金
国家重点研发计划项目(2018YFd0501102)
广东省重点领域研发计划项目(2019B020211003)。
