Construction and Expression Analysis of Exported Repteated Protein (Erp) Containing 8 Base Sequence Mutants of Mycobacterium tuberculosis | 结核分枝杆菌输出重复蛋白(Erp)8基序突变体的构建及表达分析

Chinese. 结核病(TB)是由结核分枝杆菌(Mycobacterium tuberculosis)引起的古老疾病，与艾滋病、疟疾一并成为当今世界威胁人类健康的三大传染病。输出重复蛋白(Erp)是结核分枝杆菌的一种重要毒力因子，在临床上具有一定的潜在应用价值。为了深入研究Erp的功能，本研究采用固相亚磷酰胺三酯化学方法合成包含8个PGLTS基序的基因Dom8，并利用重叠延伸PCR的方法将该片段与DomC(长度为520~855 bp)连接，经基因组装得到Erp蛋白8基序突变片段。进一步对获得的8基序全长基因进行C端、N端及CN两端缺失突变体，将其构建至原核表达载体pET-28a(+)上，将经酶切、PCR、测序鉴定的阳性质粒转化入大肠杆菌(Escherichia coli)BL21(DE3)pLysS菌株中。利用Tricine/SDS-PAGE、Western blot检测结核分枝杆菌Erp重组蛋白的表达情况。结果显示，通过重叠延伸PCR法成功扩增了erp基因PGLTS 8基序全长及8基序C-端、N-端和CN-端缺失突变体并构建至原核表达载体，经IPTG诱导后,用Tricine/SDS-PAGE得到大小分别约为13.0、17.1和6.7 kD的目的蛋白。制备弗氏佐剂疫苗免疫试验兔， ELISA测定抗体效价，血清效价达到1∶512倍时，即可心脏采血分离，制备抗血清，并对提取的蛋白进行Western blot检测，结果表明，一抗为l∶200 倍稀释的兔阳性血清，二抗为1∶800 倍稀释的HRP标记的羊抗兔IgG-HRP；Western blot检测Erp MutC8、MutN8和MutCN8，结果显示，仅目的蛋白Erp MutC8和MutN8能被结核分枝杆菌Erp抗血清识别，表明，BL21(DE3)pLysS(pET28a-C8)和BL21(DE3)pLysS(pET28a-N8)所表达目的蛋白表达特异，可以被结核分枝杆菌Erp抗血清识别，具有良好的抗原性。研究结果为深入研究Erp的功能，制备结核病的诊断抗原和基因工程重组疫苗提供基础资料。

English. Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis. It is one of the three major infectious diseases which threaten public health, the other two are AIDS(acquired immune deficiency syndrome) and malaria. Exported repeated protein (Erp) is an important virulence factors of M. tuberculosis. It has potential value in the clinical application. In order to study the function of Erp, short fragments of DomC and Dom8 synthetized by solid-phase phosphoramidite method were assembled by SOE-PCR. M. tuberculosis erp gene PGLTS length of 8 motif, C , N and CN terminal end mutantion of the 8 motif were cloned by overlap extension PCR. The clones in Escherichia coli BL21 (DE3) pLysS strain were expressed. The mutated genes were subcloned into the prokaryotic expression vector pET-28a (+). Positive plasmids were selected by enzyme digestion, PCR, and DNA sequencing. The recombinant protein expression of M. tuberculosis Erp was detected by SDS-PAGE and Western blot. The results showed that the sizes of the amplified fragments areas were as expected. The erp gene PGLTS motif length of 8 motif, C, N and CN terminal end mutant were amplified successfully by overlap extension PCR and built into the prokaryotic expression vector. Recombinant BL21 (DE3)pLysS strains was induced by IPTG. Detected by tricine/SDS-PAGE, the relative molecular weights of the expression products were 13.0, 17.1 and 6.7 kD, respectively. Prepare the Freund's adjuvant vaccine and vaccinate rabbits. The antibody titers were determined with ELISA. When the titer got 1∶512, the heart blood could be drawn and separated. After preparing the antiserum, we used Western-blot to detect the extracted proteins. The primary antibody was l∶200 dilution of rabbit positive sera. The secondary antibody was 1∶800 dilution of HRP-labeled sheep anti- rabbit IgG. The results of Western blot showed that the proteins of MutC8 and MutN8 possessed good antigenicity, which were identified by the rabbit antisera against Erp of M. tuberculosis. The experiment results showed that the target proteins of BL21 (DE3) pLysS (pET28a-C8) and BL21 (DE3) pLysS (pET28a-N8) were expressed specifically, which were identified by the rabbit antisera against Erp in Mycobacterium tuberculosis. Meanwhile, the proteins mentioned above had excellent antigenicity. The experiments provides basic data for the diagnosis of bovine tuberculosis and the recombinant vaccine antigen of the gene in the future.