鸡CVH基因截取片段的克隆、原核表达及其多克隆抗体制备-兽医专业论文.docxVIP

广西大学硕士学位论文 广西大学硕士学位论文 鸡CVH基因截取片段的克隆、原核表达及其多克隆抗体的制备 合蛋白，并制备了Cvh多克隆抗体，为PGCs鉴定和功能研究奠定了基础。 关键词：鸡CVH基因 原核表达蛋白纯化多克隆抗体 2 万方数据 广西大掌硕士掌位论文 广西大掌硕士掌位论文 鸡CVH基因截取片段的克隆、原核表达及其多克隆抗体的制备 Cloning，Prokaryotic Expression and Polyclonal antibody Preparation of Chicken CVH ABSTRACT CWrt gene is chicken VASA homolog gene，its production has ATP—dependent RNA helicase due to the protein contains DEAD·box motif．The Cvh proteins specifically exist in embryonic stem cells(ESs)and primordial germ cells(PGCs)which are necessary to evolution and differentiation of germ line cells．Thence，as a marker in germ line cells，Cvh has been used to identify and screen ESs and PGCs． The purpose of this study was to clone a part of coding sequence of CVH gene and prepare polyclonal anti·—Cvh antibody using purified His—-Cvh fusion proteins by Ni··NTA agarose matrices．Total RNA was extracted from the testis of chick at age of 3 weeks，and cDNA was obtained by RT-PCR．Encoding 2 1 3 amino acids and locating in the up-stream of CVH，and the eDNA fragment was ligated to pMD 1 8-T vector to construct recombinant plasmid pMD 1 8一T-CVH．Confirmed by sequencing，the target gene was subcloned to pET-30a vector to construct prokaryotic expression vector(pET-30a—CVH)．transformed with pET-30a—CVH，E．coli BL21(DE3)were induced to expression His-Cvh protein with 1 mmol／L Isopropyl—p-D-thiogalactopyranoside(IPTG)at 3 7℃，which Was subsequently detected by SDS—PAGE and Westem blot assays．The target protein Was purtified by Ni-NTA agarose matrices under denaturing condition(dissolved in 8 mol／L urea)．As an antigen，His—Cvh fusion protein mixed with Freund’S adjuvant and emulsificed，and subcutaneously injected New Zealand white rabbits for three times at all interval of 3 weeks． On the 7也day after the last injection，blood was collected from the heart，and serum was isolated by centrifugation．This serum was namely the polyclonal anti—Cvh antibody,and subsequently used to detect endogenous Cvh expression in the geni