【摘要】：目的構(gòu)建表達(dá)豬囊尾蚴cC1抗原的重組恥垢分枝桿菌疫苗。方法提取pET28a-cC1質(zhì)粒DNA,用xho I和BamH I雙酶切,用Klenow酶補(bǔ)平xho I酶切末端,同時(shí)提取pMV261穿梭質(zhì)粒載體,用Hind III和BamH I雙酶切,用Klenow酶補(bǔ)平Hind III酶切末端,純化后的酶切產(chǎn)物通過T4連接酶連接,構(gòu)建pMV261-cC1穿梭質(zhì)粒,測序驗(yàn)證正確后,將pMV261cC1穿梭質(zhì)粒經(jīng)電穿孔法轉(zhuǎn)化恥垢分枝桿菌,構(gòu)建重組恥垢分枝桿菌,經(jīng)熱誘導(dǎo)后,以豬囊尾蚴病患者血清為一抗進(jìn)行Western-blotting鑒定。此外,比較正常恥垢分枝桿菌和重組cC1恥垢分枝桿菌生長狀態(tài)并繪制生長曲線。結(jié)果構(gòu)建的重組穿梭載體經(jīng)酶切、測序驗(yàn)證正確。構(gòu)建的重組恥垢分枝桿菌經(jīng)熱誘導(dǎo)后,SDS-PAGE電泳分析顯示,在40 kDa處有外源性蛋白表達(dá),與預(yù)期值相符。Western-blotting顯示其可被豬囊尾蚴病患者血清識別,表明cC1抗原基因在恥垢分枝桿菌中表達(dá)成功。重組恥垢分枝桿菌與正常恥垢分枝桿菌的增殖特性無明顯差異。結(jié)論成功構(gòu)建了表達(dá)豬囊尾蚴特異性抗原cC1的重組恥垢分枝桿菌疫苗。
[Abstract]:Objective to construct a recombinant Mycobacterium pubis vaccine expressing cC1 antigen of cysticercus cellulosae. Methods pET28a-cC1 plasmid DNA, was extracted by xho I and BamH I double enzyme digestion, xho I was digested with Klenow enzyme, pMV261 shuttle plasmid vector was extracted at the same time, Hind III and BamH I were digested with Hind III and BamH I, and Hind III was digested with Klenow enzyme. The purified enzyme product was ligated by T _ 4 ligase to construct pMV261-cC1 shuttle plasmid. After sequencing, the pMV261cC1 shuttle plasmid was transformed into Mycobacterium pubis by electroporation, and the recombinant Mycobacterium pubis was constructed. After heat induction, The serum of patients with cysticercosis was identified by Western-blotting. In addition, the growth state of normal Mycobacterium pubis and recombinant cC1 was compared and the growth curve was drawn. Results the recombinant shuttle vector was digested by enzyme and sequenced. After heat induction, SDS-PAGE electrophoresis analysis showed that exogenous protein was expressed at 40 kDa, which was consistent with the expected value. Western-blotting showed that it could be recognized by serum of patients with cysticercosis. The results showed that cC1 antigen gene was successfully expressed in Mycobacterium pubis. There was no significant difference in proliferation characteristics between recombinant Mycobacterium pubis and normal Mycobacterium pubis. Conclusion Recombinant Mycobacterium pubis vaccine expressing cysticercus specific antigen cC1 was successfully constructed.
【作者單位】： 蚌埠醫(yī)學(xué)院病原生物學(xué)教研室;安徽省感染與免疫重點(diǎn)實(shí)驗(yàn)室;蚌埠醫(yī)學(xué)院免疫學(xué)教研室;
【基金】：國家自然科學(xué)基金(30600518/C030112) 安徽省高等學(xué)校省級自然科學(xué)研究重點(diǎn)項(xiàng)目(KJ2013A185)
【分類號】：R392

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