猪神经介素B受体基因慢病毒过表达载体的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2022.10.007

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (10): 3756-3763.doi: 10.16431/j.cnki.1671-7236.2022.10.007

猪神经介素B受体基因慢病毒过表达载体的构建与鉴定

邵淑玉¹, 李佳¹, 马卓^1,2, 于畅^1,2, 张莹³, 张金龙^1,2, 马志禹^1,2

1. 扬州大学兽医学院, 扬州 225009;
2. 江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009;
3. 中国农业科学院家禽研究所, 扬州 225125

收稿日期:2022-04-10 出版日期:2022-10-05 发布日期:2022-09-30
通讯作者: 张金龙,E-mail:zjl@yzu.edu.cn;马志禹,E-mail:mzy2017@yzu.edu.cn
作者简介:邵淑玉,E-mail:1463834473@qq.com;李佳,E-mail:1806157949@qq.com。
基金资助:
国家自然科学基金项目(31802149);中国博士后科学基金项目(2019M651985);江苏省自然科学基金项目(BK20180919);江苏省博士后科学基金项目(2018K207C);江苏高校优势学科建设工程资助项目(PAPD);扬州大学大学生科技创新基金项目(X20210693)

Construction and Identification of Lentiviral Overexpression Vector of Porcine Neuromedin B Receptor Gene

SHAO Shuyu¹, LI Jia¹, MA Zhuo^1,2, YU Chang^1,2, ZHANG Ying³, ZHANG Jinlong^1,2, MA Zhiyu^1,2

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;
3. Institute of Poultry, Chinese Academy of Agricultural Sciences, Yangzhou 225125, China

Received:2022-04-10 Online:2022-10-05 Published:2022-09-30

摘要/Abstract

摘要： 【目的】构建猪神经介素B受体(NMBR)基因慢病毒过表达载体,并检测其在猪Leydig细胞中的表达,为研究NMBR基因过表达在猪睾丸间质(Leydig)细胞中的功能提供参考。【方法】根据猪NMBR基因(GenBank登录号:KM058699)CDS序列设计并合成特异性酶切引物,通过RT-PCR扩增获得猪NMBR CDS全长序列,构建pMD19-T-NMBR质粒;通过Xba Ⅰ和EcoR Ⅰ对pCD513B-1和pMD19-T-NMBR质粒双酶切,构建pCD513B-1-NMBR重组质粒;将pCD513B-1-NMBR重组质粒与辅助质粒共转染人胚肾细胞(HEK-293T),转染48 h后通过荧光倒置显微镜观察细胞中绿色荧光蛋白(GFP)情况,收集细胞培养液上清获得猪NMBR基因过表达慢病毒,并通过倍比稀释法测病毒效价;然后将猪NMBR基因过表达慢病毒转染猪Leydig细胞,转染48 h后观察细胞中GFP表达,收集细胞并通过实时荧光定量PCR检测NMBR mRNA的表达情况。【结果】通过RT-PCR成功扩增获得猪NMBR CDS全长序列,大小为1 173 bp;pMD19-T-NMBR重组质粒双酶切为2条特异性条带,条带大小分别与pMD19-T质粒和猪NMBR CDS序列片段大小一致,表明成功构建pMD19-T-NMBR质粒;通过双酶切获得线性化的pCD513B-1和NMBR序列;RT-PCR和测序结果表明,成功构建慢病毒过表达载体pCD513B-1-NMBR;pCD513B-1-NMBR转染48 h后,HEK-293T细胞中呈现大量绿色荧光,表明病毒包装成功,病毒效价约为4×10⁶ TU/mL;NMBR基因慢病毒转染猪Leydig细胞48 h后,80%以上细胞有GFP表达,表明大部分细胞成功转染慢病毒;实时荧光定量PCR检测结果表明,转染慢病毒后能够极显著增加NMBR mRNA的表达(P＜0.01)。【结论】本试验成功构建了猪NMBR基因慢病毒过表达载体,并证实猪NMBR基因过表达慢病毒能够增加猪Leydig细胞NMBR基因的表达,为研究NMBR基因过表达在猪Leydig细胞中的作用奠定基础。

关键词: 神经介素B受体(NMBR); 慢病毒; 过表达; Leydig细胞

Abstract: 【Objective】 The lentiviral overexpression vector of porcine neuromedin B receptor (NMBR) gene was constructed and its expression was detected in porcine Leydig cells,in order to provide references for studying the function of NMBR gene overexpression in Leydig cells.【Method】 Specific primers were designed and synthesized according to the CDS sequence of porcine NMBR gene (GenBank accession No.:KM058699),and the full-length sequence of porcine NMBR CDS was amplified by RT-PCR.pMD19-T-NMBR was constructed.The plasmids of pCD513B-1 and pMD19-T-NMBR were digested by Xba Ⅰ and EcoR Ⅰ.pCD513B-1-NMBR plasmid was constructed.HEK-293T cells were co-transfected with pCD513B-1-NMBR and packaged plasmids,and the green fluorescence protein (GFP) was observed by fluorescence inversion microscopy after transfection for 48 h,and the porcine NMBR gene overexpression lentivirus was obtained from cell culture liquid supernatant,and the titer of the virus was measured by multiple dilution method.Then lentivirus was transfected into porcine Leydig cells,and GFP expression was observed after transfection for 48 h.The cells were collected and the expression of NMBR mRNA was detected by Real-time quantitative PCR.【Result】 The full-length sequence of porcine NMBR CDS was successfully amplified by RT-PCR,and the size was 1 173 bp.The pMD19-T-NMBR recombinant plasmid was cleaved into 2 specific bands,which were consistent with the size of pMD19-T plasmid and porcine NMBR CDS sequence fragment,respectively,indicating that pMD19-T-NMBR plasmid was successfully constructed.The linearized pCD513B-1 and NMBR sequences were obtained by double digestion.RT-PCR and sequencing results showed that the lentiviral overexpression vector pCD513B-1-NMBR was successfully constructed.48 h after pCD513B-1-NMBR transfection,HEK-293T cells showed a large amount of GFP,indicating that the packaged virus was successful,and the virus titer was about 4×10⁶ TU/mL.More than 80% of Leydig cells were expressed GFP after NMBR gene lentivirus transfection for 48 h,indicating that most of the cells were successfully transfected with lentivirus.The Real-time quantitative PCR results showed that the expression of NMBR mRNA was extremely significantly increased after transfection with lentivirus(P＜0.01).【Conclusion】 In this study,the lentivirus overexpression vector of porcine NMBR gene was successfully constructed,and it was confirmed that porcine NMBR gene overexpression lentivirus could increase the expression of NMBR gene in porcine Leydig cells.The results laid a foundation for studying the role of NMBR gene overexpression in Leydig cells.

Key words: neuromedin B receptor (NMBR); lentivirus; overexpression; Leydig cells

中图分类号:

邵淑玉, 李佳, 马卓, 于畅, 张莹, 张金龙, 马志禹. 猪神经介素B受体基因慢病毒过表达载体的构建与鉴定[J]. 中国畜牧兽医, 2022, 49(10): 3756-3763.

SHAO Shuyu, LI Jia, MA Zhuo, YU Chang, ZHANG Ying, ZHANG Jinlong, MA Zhiyu. Construction and Identification of Lentiviral Overexpression Vector of Porcine Neuromedin B Receptor Gene[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3756-3763.

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猪神经介素B受体基因慢病毒过表达载体的构建与鉴定

Construction and Identification of Lentiviral Overexpression Vector of Porcine Neuromedin B Receptor Gene

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