發(fā)布時(shí)間：2018-02-24 07:10

  本文關(guān)鍵詞： 雞CVH基因 原核表達(dá) 蛋白純化 多克隆抗體　出處：《廣西大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：CVH(Chicken VASA Homolog)基因是VASA基因的同源基因,其表達(dá)的Cvh蛋白含有DEAD-box保守基序,具有RNA解旋酶活性,在生殖系細(xì)胞中特異性存在,對(duì)配子的形成起著決定性的作用。因此,CVH基因作為生殖系細(xì)胞的標(biāo)志基因,在胚胎干細(xì)胞(ES)和原始生殖細(xì)胞(PGC)的研究中用于鑒別和篩選生殖系細(xì)胞。本研究旨在通過(guò)克隆雞CVH基因的片段,利用原核表達(dá)系統(tǒng)獲得His-Cvh融合蛋白,純化后作為抗原免疫新西蘭大白兔制備抗CVH基因的多克隆抗體。利用Trizol法提取3周齡的雞睪丸中的總RNA,用RT-PCR擴(kuò)增CVH基因中640 bp的編碼序列,并將其克隆到pMD18-T載體上構(gòu)建pMD18-T-CVH重組質(zhì)粒。經(jīng)鑒定序列正確的陽(yáng)性質(zhì)粒,將其插入的目的基因亞克隆至pET-30a表達(dá)載體上,構(gòu)建pET-30a-CVH重組質(zhì)粒,經(jīng)雙酶切鑒定正確的質(zhì)粒轉(zhuǎn)化E.coli BL21(DE3),用1 mmol/L異丙基硫代β-D-半乳糖甘(IPTG)37℃恒溫下誘導(dǎo)4hr.,使其表達(dá)目的蛋白,用SDS-PAGE與Western blot檢測(cè)和鑒定目的蛋白的表達(dá)。確定目的蛋白表達(dá)后,用8mol/L尿素溶解蛋白,通過(guò)Ni-NTA親和層析柱對(duì)His-Cvh融合蛋白進(jìn)行純化。得到的純化蛋白與弗氏佐劑混合乳化后,分三次皮下多點(diǎn)注射免疫新西蘭大白兔,每次間隔3周。于第三次注射后第7天,心臟采血后收集血清,該血清即為制備的抗CVH基因多克隆抗體。最后通過(guò)分離的第5.5天的雞胚生殖嵴作為抗原,通過(guò)Western blot檢測(cè)Cvh多克隆抗體的特異性。結(jié)果表明：(1)本實(shí)驗(yàn)克隆了雞CVH基因ORF框中的640 bp編碼片段。該序列與雞CVH基因核苷酸序列(AB004836.1)和氨基酸序列的(BAB12337.1)同源性分別為99.5%和99.2%。(2)構(gòu)建了pMD18-T-CVH重組克隆載體和pET-30a-CVH重組表達(dá)載體；在優(yōu)化條件下,pET-30a-CVH重組質(zhì)粒可在E.coli BL21(DE3)中高效表達(dá)。(3)Western blot實(shí)驗(yàn)表明,純化所得蛋白可與抗His標(biāo)簽抗體(1：5,000稀釋)特異性地結(jié)合;(4) Western blot實(shí)驗(yàn)表明,所獲得的兔源多克隆抗體(1：10,000稀釋)可與His-Cvh融合蛋白和雞胚生殖嵴中內(nèi)在的Cvh蛋白特異性的結(jié)合。本研究通過(guò)克隆雞CVH基因的部分編碼序列,利用原核表達(dá)系統(tǒng)獲得His-Cvh融合蛋白,并制備了Cvh多克隆抗體,為PGCs鑒定和功能研究奠定了基礎(chǔ)。
[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，

本文編號(hào)：1529328