鸡CVH基因截取片段的克

发布时间：2018-02-24 07:10

  本文关键词： 鸡CVH基因 原核表达 蛋白纯化 多克隆抗体　出处：《广西大学》2015年硕士论文　论文类型：学位论文

【摘要】：CVH(Chicken VASA Homolog)基因是VASA基因的同源基因,其表达的Cvh蛋白含有DEAD-box保守基序,具有RNA解旋酶活性,在生殖系细胞中特异性存在,对配子的形成起着决定性的作用。因此,CVH基因作为生殖系细胞的标志基因,在胚胎干细胞(ES)和原始生殖细胞(PGC)的研究中用于鉴别和筛选生殖系细胞。本研究旨在通过克隆鸡CVH基因的片段,利用原核表达系统获得His-Cvh融合蛋白,纯化后作为抗原免疫新西兰大白兔制备抗CVH基因的多克隆抗体。利用Trizol法提取3周龄的鸡睾丸中的总RNA,用RT-PCR扩增CVH基因中640 bp的编码序列,并将其克隆到pMD18-T载体上构建pMD18-T-CVH重组质粒。经鉴定序列正确的阳性质粒,将其插入的目的基因亚克隆至pET-30a表达载体上,构建pET-30a-CVH重组质粒,经双酶切鉴定正确的质粒转化E.coli BL21(DE3),用1 mmol/L异丙基硫代β-D-半乳糖甘(IPTG)37℃恒温下诱导4hr.,使其表达目的蛋白,用SDS-PAGE与Western blot检测和鉴定目的蛋白的表达。确定目的蛋白表达后,用8mol/L尿素溶解蛋白,通过Ni-NTA亲和层析柱对His-Cvh融合蛋白进行纯化。得到的纯化蛋白与弗氏佐剂混合乳化后,分三次皮下多点注射免疫新西兰大白兔,每次间隔3周。于第三次注射后第7天,心脏采血后收集血清,该血清即为制备的抗CVH基因多克隆抗体。最后通过分离的第5.5天的鸡胚生殖嵴作为抗原,通过Western blot检测Cvh多克隆抗体的特异性。结果表明：(1)本实验克隆了鸡CVH基因ORF框中的640 bp编码片段。该序列与鸡CVH基因核苷酸序列(AB004836.1)和氨基酸序列的(BAB12337.1)同源性分别为99.5%和99.2%。(2)构建了pMD18-T-CVH重组克隆载体和pET-30a-CVH重组表达载体；在优化条件下,pET-30a-CVH重组质粒可在E.coli BL21(DE3)中高效表达。(3)Western blot实验表明,纯化所得蛋白可与抗His标签抗体(1：5,000稀释)特异性地结合;(4) Western blot实验表明,所获得的兔源多克隆抗体(1：10,000稀释)可与His-Cvh融合蛋白和鸡胚生殖嵴中内在的Cvh蛋白特异性的结合。本研究通过克隆鸡CVH基因的部分编码序列,利用原核表达系统获得His-Cvh融合蛋白,并制备了Cvh多克隆抗体,为PGCs鉴定和功能研究奠定了基础。
[Abstract]:CVH(Chicken VASA homology gene is the homologous gene of VASA gene. The expressed Cvh protein contains DEAD-box conserved motif and has RNA helicase activity. Therefore, the CVH gene is a marker gene in reproductive cells, which plays a decisive role in the formation of gametes. The purpose of this study was to identify and screen reproductive cell lines by cloning the fragment of chicken CVH gene and to obtain His-Cvh fusion protein by prokaryotic expression system in the study of embryonic stem cell (es) and primordial germ cell (PGC). The polyclonal antibodies against CVH gene were prepared by immunizing New Zealand white rabbits as antigens. The total RNAs of three-week-old chicken testis were extracted by Trizol method, and the 640bp coding sequence of CVH gene was amplified by RT-PCR. The recombinant plasmid of pMD18-T-CVH was constructed by cloning it into pMD18-T vector. The positive plasmid with correct sequence was identified, and the target gene was subcloned into the expression vector of pET-30a to construct the recombinant plasmid of pET-30a-CVH. The recombinant plasmid was transformed into E. coli BL21 (DE3) by double enzyme digestion, and was induced by 1 mmol/L isopropyl thiothioate 尾 -Dgalactosylthioglycan at 37 鈩，

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