發(fā)布時(shí)間：2018-05-27 00:13

  本文選題：牛支原體 + 醛縮酶��； 參考：《農(nóng)業(yè)生物技術(shù)學(xué)報(bào)》2017年02期

【摘要】：牛支原體(Mycoplasma bovis,Mb)是牛(Bos taurus)的一種重要致病性支原體,其感染可引起牛的多種疾病。果糖二磷酸醛縮酶(fructose bisphosphate aldolase,FBA)是參與糖酵解過(guò)程中的關(guān)鍵酶,為3-磷酸甘油醛脫氫反應(yīng)提供底物,并廣泛存在于生物界中。為了研究FBA在Mb細(xì)胞內(nèi)的分布,本研究參照Gen Bank中Mb PG45株fba基因序列設(shè)計(jì)特異性引物,應(yīng)用PCR擴(kuò)增獲得Mb武威株fba基因并將其克隆至p MD19-T載體,在完成測(cè)序及基因優(yōu)化的基礎(chǔ)上,構(gòu)建原核表達(dá)載體p ET-fba并轉(zhuǎn)化大腸桿菌(Escherichia coli)表達(dá)菌Transetta(DE3)后經(jīng)異丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)誘導(dǎo)表達(dá),表達(dá)產(chǎn)物純化后免疫新西蘭兔(Oryctolagus cuniculus)制備多克隆抗體,并采用間接酶聯(lián)免疫吸附試驗(yàn)(enzyme linked immunosorbent assay,ELISA)測(cè)定抗體效價(jià)。進(jìn)而應(yīng)用Western blot對(duì)Mb FBA在細(xì)胞內(nèi)的分布進(jìn)行了初步的定位。結(jié)果表明,Mb武威株fba基因編碼框全長(zhǎng)876 bp(Gen Bank登錄號(hào):KX828563),編碼292個(gè)氨基酸,優(yōu)化后的基因在大腸桿菌中成功獲得表達(dá),重組蛋白r Mb FBA大小約為34 k D,主要以可溶性蛋白的形式存在;ELISA測(cè)定制備的多克隆抗體效價(jià)為1∶25 600,Western blot分析結(jié)果顯示,該蛋白具有良好的免疫原性,且在Mb胞漿及膜表面均有分布。上述結(jié)果為進(jìn)一步研究Mb FBA的生物學(xué)功能提供了理論依據(jù)。
[Abstract]:Mycoplasma bovismb) is an important pathogenic mycoplasma of Bos taurus, and its infection can cause many diseases in cattle. Fructose bisphosphate aldolase (FBA) is a key enzyme involved in glycolysis, which provides a substrate for the dehydrogenation of glyceraldehyde 3-phosphate, and is widely found in the biological world. In order to study the distribution of FBA in Mb cells, a specific primer was designed according to the fba gene sequence of Mb PG45 strain in Gen Bank. The fba gene of Mb Wuwei strain was amplified by PCR and cloned into pMD19-T vector. On the basis of complete sequencing and gene optimization, the prokaryotic expression vector p ET-fba was constructed and transformed into E. coli Escherichia coli expression strain TransettaDE3). The expression was induced by isopropyl 尾 -D-thiogalactopyranoside side ET-fba (isopropyl 尾 -D-thiogalactopyranoside). The polyclonal antibody was prepared by immunizing Oryctolagus cuniculus after purification and the titer of the antibody was determined by indirect enzyme-linked immunosorbent assay (Elisa). Furthermore, the distribution of Mb FBA in cells was preliminarily localized by Western blot. The results showed that the fba gene encoding frame of Mb Wuwei strain was 876 bp(Gen Bank accession number: KX828563G, encoding 292 amino acids. The optimized gene was successfully expressed in E. coli. The r Mb FBA of the recombinant protein was about 34 KD, and the antibody titer of polyclonal antibody determined by Elisa in the form of soluble protein was 1:25. The results of Western blot analysis showed that the protein had good immunogenicity. The distribution of Mb on the cytoplasm and on the surface of the membrane was also observed. These results provide a theoretical basis for further study of the biological function of Mb FBA.
【作者單位】： 甘肅農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(No.31360620) 甘肅省高等學(xué)�；究蒲袠I(yè)務(wù)費(fèi)項(xiàng)目
【分類號(hào)】：S852.62

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