【作者】 范雄林；

【导师】 徐志凯；

【作者基本信息】 第四军医大学 ， 病原生物学（分子微生物学）， 2001， 博士

【摘要】 结核病（tuberculosis，TB）是由结核分枝杆菌（Mycobacteriumtuberculosis，MTB）所致以呼吸道为主的慢性传染病。目前全球有1/3的人口感染MTB，每年有1000万的新发病人和300万的病人死亡，表明TB仍是影响全球人类健康的一个主要公共卫生问题。近年来，耐药结核分枝杆菌的流行、与HIV的合并感染以及人口流动的增加等因素，进一步加剧了TB对人类的威胁。卡介苗（BCG）是目前TB唯一的预防性疫苗，在发展中国家广泛应用，但其保护效果不稳定。另外，BCG接种干扰了利用PPD对MTB感染的诊断，接种免疫缺陷病人可能导致播散性感染等缺陷。鉴于BCG自身的不足，以及当前TB流行的严重性，研究更有效的疫苗具有重要意义。 基因免疫在包括TB的多种传染病的动物模型中可引起体液和细胞免疫应答。利用质粒DNA真核表达载体，已经有24个MTB抗原被表达，这些抗原主要来自MTB培养滤液蛋白（CFP）。其中有几个蛋白在动物模型中显示了较高的保护性，如Ag85复合体、ESAT6、MPT64、Hsp65和PstS3等。因而基因免疫可能是有希望的用于防治TB的一种有效方法。 MTB CFP中的蛋白质种类高达800多种。目前从中鉴定保护性抗原的方法主要是：利用生物化学的方法分离蛋白，以刺激淋巴细胞分泌细胞因子；利用TB病人血清或MTB感染鼠血清筛检MTB表达文库。 第四军医大学博十学位论文 MTB基因疫茵的研究也可作为一种潜在的研究方法，在体内用于保护性 抗原的筛选鉴定。 为了TB新型疫苗的研究，以及建立快速、有效、廉价的基因免疫 筛选保护性抗原的模式，我们采用 PCR的方法从 MTB H37Ra株基因组 中，扩增出分泌蛋白Ag85B的成熟蛋白的编码基因，用限制性内切酶消 化后，插入克隆载体 pUC中。经酶切鉴定与序列测定证实后，以亚克 隆法构建于真核表达载体pcDNA3的相应酶切位点，再用酶切鉴定证实 相应片段的正确插入。该重组子命名为pTB30m。所测定的序列用Blast 程序检索，表明 MTB H37Ra株Ag85B蛋白的编码基因与结核分枝杆菌 复合体和非结核分枝杆菌相应蛋白的基因高度同源，该序列登录入 Genbank（accession number：AF198032）。将 pTB30m以电穿子法转染 CHO 细胞，并以RTFCR法检测转染细胞中Ag85B特异的mRNA的表达， 初步证实了该质粒可在体外真核细胞中表达：另用该质粒肌注免疫 BALB／c和C57BL历小鼠，ELISA法检测免疫小鼠的体液免疫应答效果， 证实了该质粒可在体内表达。两种小鼠血清中Ag85B特异性抗体滴度分 别约为1：60和1：80。 为了检测pTB30m质粒免疫小鼠引起的细胞免疫反应，基因免疫完 成后第六周，分离免疫小鼠的脾淋巴细胞，体外用抗原再刺激，生物学 方法检测 IFN Y产生水平和 MTT法检测淋巴细胞增殖反应。pTB30m质 粒免疫的 BALB／c和 C57BU6小鼠脾细胞分泌的 IFN Y的量分别达到 1360ng／ml和 1965pg／ml，生理盐水和空质粒对照组均低于 80 pg／ml；脾 脏淋巴细胞增殖显著，刺激指数分别为3．34和4．23，生理盐水和空质粒 对照组分别为 1．39和 1．sl。 为进一步评价基因免疫的保护性，用 MTB H37RV毒株 SX fCFU 经小鼠尾静脉攻击感染基因免疫的BALB／c ，J’鼠，4W后，脾脏细菌负荷 计数。与生理盐水对照组相比，pTB30m质粒免疫BALB儿 ’J’鼠的脾脏 细菌负荷减少（0．645logl。）CFU（t＝8．584，P＜0刀05，i＝3），而空质粒对照组 仅减少（0刀57log；。）CFU（t＝l．334，P＞0．20，n＝3）。为了证实 T细胞在基因 免疫中的作用，用尼龙毛柱分离基因免疫BALB／c小鼠的T淋巴细胞， 4 第四军医人学博士学位论文 每只 5 XIO‘过继免疫正常 BALB止 ，J’鼠，同时用 MTB ＄株 10’CFU经 小鼠尾静脉攻击感染，4w后，脾脏细菌负荷计数。pTB30m质粒免疫小 鼠的T细胞过继免疫对攻击感染后抗MTB在脾脏中增殖有一定程度的 保护作用0＜0刀5，r3．534，n＝3人与生理盐水阴性对照组相比，脾脏细菌 负荷减少O．285loglOCFU。而质粒空白对照组脾脏细菌负荷减少 （0．182logl。）CFU（0．10＜P＜0二0，t＝l石37，11＝3）。 综上所述，采用编码 MTB H37Ra株 Ag85B成熟蛋白基因的真核质 粒pTB30m基因免疫，是产生抗原特异性的体液免疫反应和保护性细胞更多还原

【Abstract】 Tuberculosis(TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium fuberculosis. TB remains an important global public health problem, because about one-third of the world’s populations are infected with the M. tuberculosis and there are 10 million new cases and 3 million deaths annually, in recent years, such factors as increasing frequency of drug-resistance M tuberculosis isolates and incidence of HI V-associated tuberculosis and increasement of population movement, have aggravated further the opportunity of people worldwide face to the threat of TB. Mycobacterium bovis BCG is the only tuberculosis vaccine available for human use in many developing countries, yet its protection efficacy varies greatly. Another limitation is that it interrupts the diagnosis of TB by using purified protein derivative (PPD). Furthermore, the live BCG vaccine represents a potential health risk to immunocompromized individuals. It should be very important to develop a more effective vaccine of TB. It has been proved that the administration of DNA vaccines could generate both hwnoral and cell-mediated immune responses in many animal models of infectious diseases including TB. 24 mycobacterial antigens 6 mostly from cultural filtration proteins(CFP) of Mtuberculosis have been expressed using plasmid DNA eukaryotic vector. Among these, several antigens have showed higher protection efficacy in animal models such as Ag85 complex, ESAT6, MPT64, Hsp65 and PstS3, etc. Thus, DNA vaccination could be a promising effective solution to protect against TB. It has been showed that over 800 proteins result in the CFP of Mtuberculosis. Important methods used to identify protective antigens from CFP include, that proteins separated by biochemistry methods could be used to stimulate lymphocytes and detect kinds and quantity of cytokines secreted by lymphocytes; and that sera of patients with TB or mice infected with Mtuberculosis could be used to screen expression library of Mtuberculosis. The development of DNA vaccine of Mtuberculosis could be a potential way to identify protective antigens in vivo. In this paper, we want to develop new vaccine against TB and search for a rapid, effective and inexpensive model of DNA vaccination to screen protective antigens. The gene encoding the Ag85B mature form with two amino acids of signal sequence from genome of M tuberculosis H37Ra strain was amplified by PCR., and inserted it into cloning vector pUC 19 after restriction endonuclease digestion. Gene fragment encoding Ag85B mature protein was correctly inserted into the vector as confirmed by partial nucleotide sequencing and restriction endonuclease digestion, and then was subcloned to sites cut with HindIIl plus EcoR I of eukaryotic expression vector pcDNA3(Invitrogen Corp.) under the control of the CMV promoter. The cloning gene was correctly inserted into the vector pcDNA3 as also confirmed by restriction endonuclease digestion. This construction was called pTB3Om. Sequence of Ag85B mature protein was compared by Blast homological search via GenBank. It shows that nucleotide sequence of Ag85B mature protein of Mtuberculosis I-137Ra strain has the highest degree of homology with that of corresponds of M tuberculosis complexes and 7 higher degree of similarity with that of corresponding proteins of non-M tuberculosis. Nucleotide sequence更多还原