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

Abstract

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

CLC Number:

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

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