| 摘要: |
| 为研究绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus，JSRV)囊膜蛋白(Envelope，env)对 NIH3T3 细胞(小鼠胚胎成纤维细胞)增殖的影响，构建pCMV-dR8.91-JSRV-env慢病毒过表达载体并感染NIH3T3细胞。将JSRV-env基因连接红色荧光慢病毒报告载体pCMV-dR8.91，基因测序正确后将载体命名为pCMV-dR8.91-JSRV-env。将pCMV-dR8.91-JSRV-env重组质粒和包装辅助质粒共转染HEK 293T细胞包装产生JSRV-env重组慢病毒。将重组慢病毒悬液利用超速离心沉淀法浓缩，real-time PCR 测定病毒滴度。浓缩病毒转导NIH3T3细胞72 h后，噻唑蓝(MTT)检测细胞增殖情况。结果显示:env同源重组入pCMV-dR8.91载体，测序结果与预期序列一致；pCMV-dR8.91-JSRV-env与包装辅助质粒共转染HEK 293T细胞72 h，real-time PCR验证JSRV-env过表达极显著(P<0.01)，JSRV-env重组慢病毒平均滴度为2.99×108 IU/mL；MTT分析显示10 μg JSR-env慢病毒感染NIH3T3细胞72 h显著促进NIH3T3细胞增殖(P<0.05)。综上，成功构建了JSRV-env慢病毒过表达载体，证实JSRV-env能够促进NIH3T3细胞增殖。 |
| 关键词: 绵羊肺腺瘤病毒 囊膜基因 慢病毒 细胞增殖 致病机制 |
| DOI：10.11841/j.issn.1007-4333.2020.07.12 |
| 投稿时间：2019-11-20 |
| 基金项目:国家自然科学基金项目(31360597,31760721)；内蒙古草原英才创新团队项目(20151031)；内蒙古科技应用研究项目(2019GG240) |
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| Construction of JSRV-env lentiviral vector and its influence on proliferation of NIH3T3 cells |
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YANG Hui,LIU Shuying
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| (College of Veterinary Medicine/Key Laboratory of Basic Veterinary Science/Key Laboratory of Clinical Diagnosis and Treatment Technology inAnimal Disease of Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot 010018, China) |
| Abstract: |
| To study the effect of the envelope(env)of Jaagsiekte sheep retrovirus(JSRV)on the proliferation of NIH3T3 cells(mouse embryo fibroblast cells), pCMV-dR8. 91-JSRV-env lentiviral overexpression vector was constructed and infected with NIH3T3 cells. The JSRV-env gene was ligated into the red fluorescent lentiviral reporter vector pCMV-dR8. 91, and the gene was correctly sequenced and named pCMV-dR8. 91-JSRV-env. The pCMV-dR8. 91-JSRV-env recombinant plasmid and packaging helper plasmid were co-transfected into HEK 293T cell packaging to produce recombinant JSRV-env lentivirus. The recombinant lentivirus suspension was concentrated by ultracentrifugation, and the virus titer was determined by real-time PCR. After the concentrated virus transduced NIH3T3 cells for 72 h, thiazole blue(MTT)was used to detect cell proliferation. The results showed that: env gene was completely and correctly recombined into the pCMV-dR8. 91 vector by sequencing; pCMV-dR8. 91-JSRV-env was co-transfected with packaging helper plasmids into 293T cells for 72 h. Real-time PCR verified that JSRV-env was very significantly overexpressed(P<0. 01)and the average titer of JSRV-env lentivirus was 2. 99×108 IU/mL; MTT analysis showed that infection of NIH3T3 cells by 10 μg JSRV-env lentivirus for 72 h significantly promoted the proliferation of NIH3T3 cells(P<0. 05). In conclusion, the JSRV-env lentiviral overexpression vector was successfully constructed, and JSRV-env can promote the proliferation of NIH3T3 cells. |
| Key words: Jaagsiekte sheep retrovirus envelope protein lentivirus cell proliferation pathogenic mechanism |
