原核表达的结核分枝杆菌H_（37）RV株重组ESAT-6和CFP-10蛋白的功能分析
摘要
结核病(Tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)引起的高度传染性疾病。目前,抗结核最有效的疫苗是卡介苗(BCG)。与致病性的结核杆菌相比,BCG缺失了很多基因,比如,编码ESAT-6和CFP-10蛋白的基因RV3875和RV3874。ESAT-6和CFP-10是结核杆菌重要的分泌性抗原,同时,也是结核杆菌的毒力因子。研究这ESAT-6和CFP-10蛋白的功能,有助于我们设计新的抗结核疫苗和诊断试剂。
利用分子生物学的手段获得了编码ESAT-6和CFP-10的基因,并且成功的在原核表达系统中表达了ESAT-6和CFP-10蛋白(分别命名为rESAT-6和rCFP-10)。通过镍亲和层析的方法纯化rESAT-6和rCFP-10,再经过Triton X-114以及Q sepharose阴离子交换树脂对rESAT-6和rCFP-10中残留的内毒素进行去除。通过免疫家兔获得抗rESAT-6和rCFP-10的血清。在蛋白的初步功能实验中,rESAT-6和rCFP-10能抑制LPS诱导的巨噬细胞分泌的肿瘤坏死因子α(TNF-α)的表达,并且rESAT-6还能明显的抑制放线菌素D (Actinomycin D, ActD)杀伤L929细胞的作用,这些结果表明rESAT-6和rCFP-10可能抑制机体的固有免疫。本研究有助于对ESAT-6和CFP-10蛋白功能的进一步了解,并为以ESAT-6和CFP-10为靶点的抗结核疫苗的设计提供了新的思路。
Tuberculosis (TB) was caused by the pathogenic mycobacterium tuberculosis (MTB) which remains the single largest infectious disease with high mortality in humans, leading to 3 million deaths and approximately 8-10 million people are infected annually. Out of the total number of cases, 40 percent of cases are accommodated in South and East Asia. According to WHO estimates, China has the world's second largest tuberculosis epidemic, behind only India, with more than 1.3 million new cases of tuberculosis every year.
Cell-mediated immunity plays a key role in host protection against TB. In particular, gamma interferon (IFN-γ)-secreting T cells have been shown to be important for the protective immune response.
The only currently available vaccine against TB is the attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG). The efficacy of this vaccine varies from 0 to 80% in different populations.
Comparative genomic analysis between M.tuberculosis and Mycobacterium bovis Bacille Calmette-Guérin (BCG), the attenuated vaccine strain, has revealed that 16 chromosomal regions of deletion (RD) exist in the latter. Among these, the RD1 is the only region absent in all strains of BCG and plays a dominant role in virulence of TB. This region contains nine protein-coding genes and two of these (Rv3874 and Rv3875), coding for the secreted proteins CFP-10 and ESAT-6. CFP-10 and ESAT-6 were also found to be a strong target for human B- and T-cell responses, and these antigens induced both high levels of IFN-γwhich could activate macrophage and enhance the effect of macrophage on the growth inhibition and killing of MTB. So there two proteins could be as potential candidate antigens for developing vaccines and diagnostic tools. On the other hand, ESAT-6 and CFP-10 also play an essential role in tuberculosis pathogenesis. They can deactivate the function of macrophage, for example, reduce the ROS level, decline the expression of inflammatory factors and inhibit the fusion of phagosome-lysosome.
In this paper, the genes encoding ESAT-6 and CFP-10 protein were amplified PCR from the genome of M. tuberculosis H37Rv strain. ESAT-6 and CFP-10 genes were constructed into the prokaryotic expression vector pET28a by recombinant DNA techniques and were identified by enzymatic digestion and sequence analysis. ESAT-6 and CFP-10 proteins containing the C-terminal His-tag (rESAT-6 and rCFP-10) were induced expression by IPTG in E.coli BL21 (DE3), in partially soluble form. We obtained the supernatant containing rESAT-6 and rCFP-10 by using ultrasonic techniques, subsequently, the supernatant was loaded onto nickel affinity column, rESAT-6 and rCFP-10 with His-tag could bind to the resin, non-specific proteins were removed, then, and the eluted fractions from affinity chromatography were applied to G-25 gel filtration to remove the small molecular salts. It necessary to remove endotoxin from rESAT-6 and rCFP-10 for cell based assay. There are two alternative methods for removing endotoxin, one is use the 1% (volume ratio) nonionic detergent Triton X-114 in washing buffer of NI+ affinity column to remove endotoxin; the other is based on a Q- Sepharose matrix, rESAT-6 and rCFP-10 were eluted once the salt concentration has reached 300 mM NaCl. Endotoxin on the other hand does not come off the matrix until about 1 M NaCl. At last, we got the purified rESAT-6 and rCFP-10 successfully.
Following, we used a method from a paper to form an ESAT-6/CFP-10 complex in vitro, but the folding result is disappointment, impling that the method is not suitable for rESAT-6 and rCFP-10 with C teminal His-tag. We harvested the antisera by immunized the rabbit with rESAT-6 and rCFP-10, and the sera can be used to detect the ESAT-6 or CFP-10 protein existing in the blood of human or animal. Meanwhile, we could use the antibody to block the effect of rESAT-6 and rCFP-10 in cell based assay.
The biological functions of rESAT-6 and rCFP-10 were investigated. rESAT-6 and rCFP-10 could inhibit the growth of RAW264.7, L929 and mouse splenocyte, especially when the concentration of proteins is above 20 mg/L. rESAT-6 and rCFP-10 with certain concentration were added into the RAW264.7 culture medium, then we detected the level of TNF-αby L929 killing assay and RT-PCR, The results showed that rESAT-6 and rCFP-10 can inhibit the expression of TNF-αon the LPS-stimulated macrophage, and rESAT-6 not rCFP-10 seems inhibiting the IFN-γbasal expression of RAW264.7 cell. In L929 killing assay, we have used actinomycin D (actD) to sensitize the L929 cell to TNF-α.
ActD is an inhibitor of RNA synthesis and acts as a potent inducer of apoptosis in several cell lines containing the cell line L929. Accidently, we found that the growth behavior of L929 cell incubated with actD in the presence of rESAT-6 was better than absence of the protein. After optimizating assay conditions, we confirmed the rESAT-6 can antagonize the effect of actD again, and the most effective concentration of protein is 40 mg/L. Finally, rESAT-6 and rCFP-10 fail to induce proliferation of normal mouse splenocyte.
In summary, the ESAT-6 and CFP-10 genes were isolated, and rESAT-6 and rCFP-10 were expressed and purified successfully. Harvest the antisera by immunizing rabbits with rESAT-6 and rCFP-10 for the later assay. In cell based assay, we have found that rESAT-6 and rCFP-10 can inhibit the expression of TNF-αon the LPS-stimulated macrophage.ESAT-6 not CFP-10 protein can antagonize the effect of actD. Seemlying, in the next plan, we need to search for the mechanism of ESAT-6 and apoptosis, and can develop tuberculosis vaccine and diagnostic kit on the base of ESAT-6 and CFP-10.
利用分子生物学的手段获得了编码ESAT-6和CFP-10的基因,并且成功的在原核表达系统中表达了ESAT-6和CFP-10蛋白(分别命名为rESAT-6和rCFP-10)。通过镍亲和层析的方法纯化rESAT-6和rCFP-10,再经过Triton X-114以及Q sepharose阴离子交换树脂对rESAT-6和rCFP-10中残留的内毒素进行去除。通过免疫家兔获得抗rESAT-6和rCFP-10的血清。在蛋白的初步功能实验中,rESAT-6和rCFP-10能抑制LPS诱导的巨噬细胞分泌的肿瘤坏死因子α(TNF-α)的表达,并且rESAT-6还能明显的抑制放线菌素D (Actinomycin D, ActD)杀伤L929细胞的作用,这些结果表明rESAT-6和rCFP-10可能抑制机体的固有免疫。本研究有助于对ESAT-6和CFP-10蛋白功能的进一步了解,并为以ESAT-6和CFP-10为靶点的抗结核疫苗的设计提供了新的思路。
Tuberculosis (TB) was caused by the pathogenic mycobacterium tuberculosis (MTB) which remains the single largest infectious disease with high mortality in humans, leading to 3 million deaths and approximately 8-10 million people are infected annually. Out of the total number of cases, 40 percent of cases are accommodated in South and East Asia. According to WHO estimates, China has the world's second largest tuberculosis epidemic, behind only India, with more than 1.3 million new cases of tuberculosis every year.
Cell-mediated immunity plays a key role in host protection against TB. In particular, gamma interferon (IFN-γ)-secreting T cells have been shown to be important for the protective immune response.
The only currently available vaccine against TB is the attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG). The efficacy of this vaccine varies from 0 to 80% in different populations.
Comparative genomic analysis between M.tuberculosis and Mycobacterium bovis Bacille Calmette-Guérin (BCG), the attenuated vaccine strain, has revealed that 16 chromosomal regions of deletion (RD) exist in the latter. Among these, the RD1 is the only region absent in all strains of BCG and plays a dominant role in virulence of TB. This region contains nine protein-coding genes and two of these (Rv3874 and Rv3875), coding for the secreted proteins CFP-10 and ESAT-6. CFP-10 and ESAT-6 were also found to be a strong target for human B- and T-cell responses, and these antigens induced both high levels of IFN-γwhich could activate macrophage and enhance the effect of macrophage on the growth inhibition and killing of MTB. So there two proteins could be as potential candidate antigens for developing vaccines and diagnostic tools. On the other hand, ESAT-6 and CFP-10 also play an essential role in tuberculosis pathogenesis. They can deactivate the function of macrophage, for example, reduce the ROS level, decline the expression of inflammatory factors and inhibit the fusion of phagosome-lysosome.
In this paper, the genes encoding ESAT-6 and CFP-10 protein were amplified PCR from the genome of M. tuberculosis H37Rv strain. ESAT-6 and CFP-10 genes were constructed into the prokaryotic expression vector pET28a by recombinant DNA techniques and were identified by enzymatic digestion and sequence analysis. ESAT-6 and CFP-10 proteins containing the C-terminal His-tag (rESAT-6 and rCFP-10) were induced expression by IPTG in E.coli BL21 (DE3), in partially soluble form. We obtained the supernatant containing rESAT-6 and rCFP-10 by using ultrasonic techniques, subsequently, the supernatant was loaded onto nickel affinity column, rESAT-6 and rCFP-10 with His-tag could bind to the resin, non-specific proteins were removed, then, and the eluted fractions from affinity chromatography were applied to G-25 gel filtration to remove the small molecular salts. It necessary to remove endotoxin from rESAT-6 and rCFP-10 for cell based assay. There are two alternative methods for removing endotoxin, one is use the 1% (volume ratio) nonionic detergent Triton X-114 in washing buffer of NI+ affinity column to remove endotoxin; the other is based on a Q- Sepharose matrix, rESAT-6 and rCFP-10 were eluted once the salt concentration has reached 300 mM NaCl. Endotoxin on the other hand does not come off the matrix until about 1 M NaCl. At last, we got the purified rESAT-6 and rCFP-10 successfully.
Following, we used a method from a paper to form an ESAT-6/CFP-10 complex in vitro, but the folding result is disappointment, impling that the method is not suitable for rESAT-6 and rCFP-10 with C teminal His-tag. We harvested the antisera by immunized the rabbit with rESAT-6 and rCFP-10, and the sera can be used to detect the ESAT-6 or CFP-10 protein existing in the blood of human or animal. Meanwhile, we could use the antibody to block the effect of rESAT-6 and rCFP-10 in cell based assay.
The biological functions of rESAT-6 and rCFP-10 were investigated. rESAT-6 and rCFP-10 could inhibit the growth of RAW264.7, L929 and mouse splenocyte, especially when the concentration of proteins is above 20 mg/L. rESAT-6 and rCFP-10 with certain concentration were added into the RAW264.7 culture medium, then we detected the level of TNF-αby L929 killing assay and RT-PCR, The results showed that rESAT-6 and rCFP-10 can inhibit the expression of TNF-αon the LPS-stimulated macrophage, and rESAT-6 not rCFP-10 seems inhibiting the IFN-γbasal expression of RAW264.7 cell. In L929 killing assay, we have used actinomycin D (actD) to sensitize the L929 cell to TNF-α.
ActD is an inhibitor of RNA synthesis and acts as a potent inducer of apoptosis in several cell lines containing the cell line L929. Accidently, we found that the growth behavior of L929 cell incubated with actD in the presence of rESAT-6 was better than absence of the protein. After optimizating assay conditions, we confirmed the rESAT-6 can antagonize the effect of actD again, and the most effective concentration of protein is 40 mg/L. Finally, rESAT-6 and rCFP-10 fail to induce proliferation of normal mouse splenocyte.
In summary, the ESAT-6 and CFP-10 genes were isolated, and rESAT-6 and rCFP-10 were expressed and purified successfully. Harvest the antisera by immunizing rabbits with rESAT-6 and rCFP-10 for the later assay. In cell based assay, we have found that rESAT-6 and rCFP-10 can inhibit the expression of TNF-αon the LPS-stimulated macrophage.ESAT-6 not CFP-10 protein can antagonize the effect of actD. Seemlying, in the next plan, we need to search for the mechanism of ESAT-6 and apoptosis, and can develop tuberculosis vaccine and diagnostic kit on the base of ESAT-6 and CFP-10.
引文
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