Construction and expression of a prokaryotic expression vector for the goat sry gene Wang Xiao1, Guang-Xin E1, Cheng Shu-Zhu1, Ni Wei-Wei1, Ma Yue-Hui2, Chu Ming-Xing2, Huang Yong-Fu1,* College of Animal Science and Technology, Chongqing Key Laboratory of Forage and Herbivore, Chongqing Engineering Research Centre for Herbivores Resource Protection and Utilization, South west University, Chongqing-400 716, China 1College of Animal Science and Technology, Chongqing Key Laboratory of Forage and Herbivore, Chongqing Engineering Research Centre for Herbivores Resource Protection and Utilization, South west University, Chongqing, 400716, China 2Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing-100 193, China *Corresponding author's e-mail: H67738337@swu.edu.cn
Abstract Goats are economically important animals in the world and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl β-D-Thiogalactoside (IPTG), the cells were lysed and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1: 50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future. Top Keywords Goat, Prokaryotic expression vector, SRY gene. Top |