本文選題：旋毛蟲 + 原核表達(dá)�。� 參考：《中國獸醫(yī)學(xué)報(bào)》2017年01期

【摘要】：利用RT-PCR技術(shù)從旋毛蟲成蟲得到T3223-7基因,并進(jìn)行擴(kuò)增克隆到原核克隆載體pMD18-T中,將重組質(zhì)粒轉(zhuǎn)入克隆菌DH5α,提取質(zhì)粒進(jìn)行酶切和測序鑒定,連接至pET-28a表達(dá)載體,最后轉(zhuǎn)入表達(dá)菌Rosetta(DE3)。用1mmol/L IPTG誘導(dǎo)培養(yǎng)重組表達(dá)菌,對(duì)菌體裂解物進(jìn)行SDS-PAGE分析,發(fā)現(xiàn)重組蛋白以包涵體的形式表達(dá),約為40 000,與理論值相符。將純化后的重組蛋白免疫家兔,制備兔抗T3223-7蛋白多克隆抗體,間接ELISA測定多抗效價(jià)達(dá)1∶320 000,Western blot檢測表明制備的多克隆抗體能與T3223-7抗原發(fā)生特異性反應(yīng),并能識(shí)別和結(jié)合旋毛蟲成蟲排泄分泌物(ES)。
[Abstract]:T3223-7 gene was obtained from adult Trichinella spiralis by RT-PCR technique, and cloned into prokaryotic clone vector pMD18-T. The recombinant plasmid was transferred to DH5 偽. The recombinant plasmid was digested and sequenced and ligated to pET-28a expression vector. Finally, the recombinant plasmid was transferred into the expression strain Rosetta-DE3. The recombinant expression bacteria were induced by 1mmol/L IPTG and analyzed by SDS-PAGE. It was found that the recombinant protein expressed in the form of inclusion body was about 40 000, which was consistent with the theoretical value. Rabbit anti-T3223-7 polyclonal antibody was prepared by immunizing rabbits with purified recombinant protein. Indirect ELISA assay showed that the polyclonal antibody could react specifically with T3223-7 antigen. It can recognize and bind the excretory secretion of Trichinella spiralis.
【作者單位】： 吉林大學(xué)人獸共患病研究所/人獸共患病研究教育部重點(diǎn)實(shí)驗(yàn)室;中國疾病預(yù)防控制中心寄生蟲病預(yù)防控制所;河南科技大學(xué)動(dòng)物疫病與公共安全重點(diǎn)實(shí)驗(yàn)室;
【基金】：國際(地區(qū))合作與交流項(xiàng)目(31520103916) 國家自然科學(xué)基金資助項(xiàng)目(31402185)
【分類號(hào)】：S852.7
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本文編號(hào)：1887704