梅毒螺旋体TpN17重组抗原的表达、纯化及鉴定

【作者】 刘娟；

【导师】 陈朝银；

【作者基本信息】 昆明理工大学 ， 生物化工， 2006， 硕士

【摘要】 梅毒是由梅毒螺旋体（Treponema pallidum,Tp）引起的一种危害性极大的全世界流行的性传播疾病。血清学试验以其快速、简便、经济等优点在临床检测中得到广泛的应用,但是,目前的检测方法依然存在着灵敏性差、假阳性率高、抗原来源相对匮乏等缺点,而采用基因工程抗原代替天然抗原建立的酶联免疫吸附试验（ELISA）在血清学检测中显示了良好的应用前景。本研究旨在通过基因工程技术制备梅毒螺旋体TpN17重组脂蛋白抗原,为进一步研究开发临床检测效果更好的新型梅毒酶联免疫诊断试剂盒提供实验材料。 首先,我们通过PCR的方法从Tp基因组中扩增编码TpN17成熟肽的目的基因片段,经HindⅢ和EcoRI双酶切后,定向克隆至pET-28b（+）中,构建原核表达载体pET-28b（+）/tpn17,转化至E.coli BL21（DE3）中,通过菌体PCR、酶切反应及进一步测序筛选获得了工程菌株BL21/pET-28b（+）/tpn17。工程菌经37℃培养,1.0mmol/L IPTG诱导4h后可获得高表达量的重组蛋白,SDS-PAGE分析显示其分子量约为19kD,与His-TpN17融合蛋白理论分子量值（18.7kD）相符,凝胶扫描分析表明诱导后目的蛋白His-TpN17产量约为菌体蛋白总量的31%。 进一步的研究发现,His-TpN17重组蛋白主要以可溶性形式存在于大肠杆菌胞内。纯化过程中我们发现,反复冻融加溶菌酶破碎方法的联合采用以及对破碎后菌体沉淀进行多次洗涤等改进方案可以使目的蛋白更多地释放到可溶性上清粗提液中,经Ni²⁺螯合层析纯化后,重组蛋白的SDS-PAGE仅在约19kD处显示单一蛋白条带,扫描分析其纯度在95%以上,Western-blot进一步证实该条带确为His-TpN17蛋白纯化后产物,并且该重组抗原与二期梅毒血清发生了很强的免疫反应。更多还原

【Abstract】 Syphilis is a very harmful sexually transmitted disease of humans over the world that is caused by infection with the bacteria Treponema pallidum subsp. pallidum （T. pallidum）. Serological test of syphilis has the advantages of high-speed, easily handling and low cost, and has been used in clinica extensively. However, the current serological testing assay of syphilis does not present the satisfied sensitivity and specificity. Enzyme-linked immunosorbent assay （ELISA） based on genetic engineering antigen instead of natural antigen would provide the solution to improve the sensitivity and specificity in serological examination. In this study, we produced a recombinant lipoprotein antigen designated TpN17 by genetic engineering technique. This protein could be used to as a key antigen in the development of ELISA diagnostic kit of syphilis.First of all, in this study, we cloned target gene encoding mature peptide TpN17 from Tp genome by using polymerase chain reaction （PCR） technique. Then, the gene was inserted into vector by restriction enzyme HindIII and EcoRI digestion. The construct was tansformed into the competent E.coli BL21 （DE3 ） for expression. The recombinants were screened by antibiotic Kanamycin plate, then it was confirmed by PCR, restriction enzyme digestion and DNA sequencing. Under the induction with 1.0mmol/L IPTG at 37℃ for 4 hours, the engineering strain BL21/pET-28b（+）/tpnl7 could produce large amount of recombinant protein. SDS-PAGE analysis indicated that the molecular weight of the recombinant protein was about 19kD, which is very close to the molecular weight of His-TpN17 fusion protein（18.7kD ）. Scanning analysis indicated that the induced target protein （His-TpN17 fusion protein ） accounts for about 31% of the total bacterial proteins.Secondly, we found that His-TpN17 fusion protein was expressed primarily in the form of soluble supernatant intracellularly in E.coli. In the process of purification for His-TpN17 fusion protein, more soluble target protein could be obtained by using repeat freeze-thaw with lysozyme and washing bacteria cellular debris repeatedly. In addition, SDS-PAGE indicated that the molecular weight of recombinant protein purified by Ni²⁺ chelate chromatography was about 19kD and its purity reached above 95%. Western-blot further confirmed that the purified product was TpN17 recombinant antigen because it could react specifically with human secondary syphilitic sera. In conclusion, wesuccessfully cloned the gene of TpN17 lipoprotein and produced the recombinant in E.coli system. The purified recombinant of TpN17 protein could be used as a key antigen in the manufacturing of ELISA diagnostic kit of syphilis.更多还原