發(fā)布時間：2018-05-23 21:08

  本文選題：白紋伊蚊 + Ab-ADGF-B基因��； 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:獲取白紋伊蚊ADGF-B(Ab-ADGF-B)活性蛋白,探討其可能的生物學(xué)功能。方法:從NCBI數(shù)據(jù)庫中下載白紋伊蚊Ab-ADGF-B基因ORF序列(GenBank No.AAV90660),對基因序列進(jìn)行生物信息學(xué)分析;將其克隆到原核表達(dá)載體pET32a(+)中,將測序正確的重組質(zhì)粒pET32a(+)Ab-ADGF-B(rAb-ADGF-B)轉(zhuǎn)化入表達(dá)菌Rosetta-gamiB(DE3)中,經(jīng)異丙基-β-D硫代半乳糖苷(IPTG)誘導(dǎo)表達(dá)并用SDS-PAGE和免疫印跡(western blotting)鑒定。用Ni-NTA親和層析柱純化rAb-ADGF-B蛋白后制備抗血清,采用間接酶聯(lián)免疫吸附法(ELISA)測抗體效價。采用分光光度法檢測rAb-ADGF-B蛋白的ADA酶催化活性;同時采用細(xì)胞計數(shù)法檢測rAb-ADGF-B對小鼠巨噬細(xì)胞(ANA)的增殖作用;最后檢測rAb-ADGF-B對人全血部分凝血酶原時間(prothrombin time,PT),凝血酶時間(thrombin time,TT)及凝血活酶時間(activated partial thromboplastin time,APTT)的影響。結(jié)果:1.成功構(gòu)建rAb-ADGF-B重組質(zhì)粒;測序結(jié)果顯示:成熟肽基因長1545 bp,編碼514個氨基酸;結(jié)構(gòu)域預(yù)測顯示具有1個ADGF結(jié)構(gòu)域。在20℃、150rpm的條件下經(jīng)0.4 m M的IPTG誘導(dǎo)24 h獲得可溶性rAb-ADGF-B蛋白。獲得兔抗rAb-ADGF-B多克隆抗體,效價為1:10000。Western blotting證實獲取的重組蛋白是目的蛋白。在30℃,pH 7.0條件下,0.67μM rAb-ADGF-B可以催化100μM腺苷生成肌苷;同時,0.134μM rAb-ADGF-B蛋白和ANA細(xì)胞共培養(yǎng)36 h后,細(xì)胞數(shù)量約由5×104增加到14×105。此外,2.67μM rAb-ADGF-B作用下,PT、TT和APTT分別為13.33S、15.23S、和35S,無延時效應(yīng)。結(jié)論:成功獲取白紋伊蚊唾液腺源rAb-ADGF-B可溶性蛋白,該蛋白具有ADA酶活性,對小鼠巨噬細(xì)胞增殖有影響。
[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，

本文編號：1926405