白纹伊蚊ADGF-B基因克隆表达及生物学功能初探

发布时间：2018-05-23 21:08

  本文选题：白纹伊蚊 + Ab-ADGF-B基因　； 参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:获取白纹伊蚊ADGF-B(Ab-ADGF-B)活性蛋白,探讨其可能的生物学功能。方法:从NCBI数据库中下载白纹伊蚊Ab-ADGF-B基因ORF序列(GenBank No.AAV90660),对基因序列进行生物信息学分析;将其克隆到原核表达载体pET32a(+)中,将测序正确的重组质粒pET32a(+)Ab-ADGF-B(rAb-ADGF-B)转化入表达菌Rosetta-gamiB(DE3)中,经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达并用SDS-PAGE和免疫印迹(western blotting)鉴定。用Ni-NTA亲和层析柱纯化rAb-ADGF-B蛋白后制备抗血清,采用间接酶联免疫吸附法(ELISA)测抗体效价。采用分光光度法检测rAb-ADGF-B蛋白的ADA酶催化活性;同时采用细胞计数法检测rAb-ADGF-B对小鼠巨噬细胞(ANA)的增殖作用;最后检测rAb-ADGF-B对人全血部分凝血酶原时间(prothrombin time,PT),凝血酶时间(thrombin time,TT)及凝血活酶时间(activated partial thromboplastin time,APTT)的影响。结果:1.成功构建rAb-ADGF-B重组质粒;测序结果显示:成熟肽基因长1545 bp,编码514个氨基酸;结构域预测显示具有1个ADGF结构域。在20℃、150rpm的条件下经0.4 m M的IPTG诱导24 h获得可溶性rAb-ADGF-B蛋白。获得兔抗rAb-ADGF-B多克隆抗体,效价为1:10000。Western blotting证实获取的重组蛋白是目的蛋白。在30℃,pH 7.0条件下,0.67μM rAb-ADGF-B可以催化100μM腺苷生成肌苷;同时,0.134μM rAb-ADGF-B蛋白和ANA细胞共培养36 h后,细胞数量约由5×104增加到14×105。此外,2.67μM rAb-ADGF-B作用下,PT、TT和APTT分别为13.33S、15.23S、和35S,无延时效应。结论:成功获取白纹伊蚊唾液腺源rAb-ADGF-B可溶性蛋白,该蛋白具有ADA酶活性,对小鼠巨噬细胞增殖有影响。
[Abstract]:Objective: to obtain the active protein of Aedes albopictus ADGF-B and to explore its possible biological function. Methods: the Ab-ADGF-B gene ORF sequence of Aedes albopictus (Aedes albopictus) was downloaded from NCBI database, and the gene sequence was analyzed by bioinformatics, and cloned into prokaryotic expression vector pET32a (pET32a). The expression was induced by isopropyl- 尾 -D-galactothioglycoside (IPTG) and identified by SDS-PAGE and Western blotting. The antiserum of rAb-ADGF-B protein was purified by Ni-NTA affinity chromatography and the antibody titer was measured by indirect enzyme-linked immunosorbent assay (Elisa). The catalytic activity of rAb-ADGF-B protein was detected by spectrophotometry, and the proliferation of rAb-ADGF-B on murine macrophages was detected by cell count method. Finally, the effects of rAb-ADGF-B on prothrombin time, thrombin time and activated partial thromboplastin time of human whole blood were detected. The result is 1: 1. The recombinant plasmid of rAb-ADGF-B was successfully constructed, and the result of sequencing showed that the mature peptide gene was 1545 BP long, encoding 514 amino acids, and the domain prediction showed that the mature peptide gene had a ADGF domain. The soluble rAb-ADGF-B protein was induced by 0.4 mm IPTG at 20 鈩，

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