背景与目的：结核病（tuberculosis，TB）是由结核分枝杆菌（mycobacterium tuberculosis，MTB）感染所引起的传染性疾病。近年来，随着耐药性结核（drug resistant TB，DR-TB）的流行以及MTB与HIV共感染的情况逐年增加，尤其是缺少对活动性结核和MTB潜伏感染（latent mycobacterium tuberculosis infections，LTBI）敏感特异的诊断方法，进一步增加了TB的防控难度。如何实现对TB的早期诊断、规范治疗以及控制DR-TB，仍是全球TB预防与控制研究中的热点和难点问题。结核菌素皮肤试验（tuberculin skin test，TST）是目前应用最广的一种细胞免疫诊断方法，但其检测结果存在较高的假阳性率，亟需寻找新的MTB特异性抗原用于TST检测。培养滤液蛋白10（culture filtrate protein，CFP10）、早期分泌性抗原性靶蛋白6（early secreting antigen target 6，ESAT6）、Rv2654c和Rv2645蛋白是近年来所发现的在人致病MTB菌株表达的特异蛋白，而在卡介苗（Bacillus Calmette-Guérin，BCG）菌株中不表达，但上述优势抗原在TST检测中的应用价值尚未明确。

材料与方法：采用基因工程技术分别构建含MTB的ESAT6-CFP10（rEC）、Rv2654c及Rv2645基因的pET-28a重组表达质粒，并用限制性内切酶酶切和直接测序的方法，确定重组质粒序列正确。将rEC、Rv2654c及Rv2645重组质粒转化入大肠杆菌BL21（DE3）中，通过表达条件优化，确定以0.1 mmol/L IPTG、30℃条件诱导重组蛋白表达，并采用亲和Ni 柱层析、阴离子交换CaptoQ层析、分子筛Superdex 200层析等方法纯化重组蛋白。通过Western blot及质谱验证目标蛋白的正确性。以结核菌素纯蛋白衍生物（purified protein derivative of tuberculin，TB-PPD）作为参考对重组蛋白进行效力测定。并构建MTB活菌、MTB灭活菌体及BCG致敏的DTH豚鼠模型，以TB-PPD作为阳性对照，将重组蛋白rEC、Rv2654c、Rv2645单一或联合注射于三组豚鼠模型皮内，于注射后12、24、48 h记录局部红晕/硬结的横、纵直径，分别将12~24 h、24~48 h直径平均值进行比较分析，评估DTH强度。同时将重组蛋白诱导三组致敏豚鼠的DTH结果与TB-PPD的结果进行比较。

研究结果：经双酶切、琼脂糖凝胶电泳及测序鉴定，结果显示pET-28a重组表达质粒中rEC、Rv2654c、Rv2645基因序列与已知目的基因序列一致，重组表达质粒构建成功。将上述重组质粒转化至大肠杆菌BL21（DE3）中并对其进行优化诱导表达，经SDS-PAGE凝胶电泳分析，结果表明各重组蛋白均以可溶性蛋白形式表达。经亲和Ni 柱层析、阴离子交换层析、分子筛层析等方法，分别获得高纯度重组蛋白rEC、Rv2654c、Rv2645（纯度＞95%）；Western Blot实验证实rEC、Rv2654c、Rv2645蛋白均与预期分子量26.5 kDa、6.5 kDa、16.7 kDa一致，蛋白质谱分析结果表明重组蛋白序列正确。重组蛋白效力测定结果表明，5.0 μg/mL rEC、10.0 μg/mL Rv2654c、10.0 μg/mL Rv2645的重组蛋白与1.0 μg/mL TB-PPD的皮试结果基本一致。免疫功能实验结果发现，在重组蛋白诱导MTB活菌致敏豚鼠的DTH试验中，rEC（5.0 μg/mL）、Rv2654c（10.0 μg/mL）、Rv2645（10.0 μg/mL）以及[rEC（3.3 μg/mL）+Rv2654c（3.3 μg/mL）+Rv2645（3.3 μg/mL）]诱导产生的红晕/硬结，12~24 h的直径平均值分别为12.3mm、11.8mm、12.3mm、15.8mm，24~48 h的直径平均值分别为9.3mm、8.8mm、8.7mm、13.0mm，结果均为阳性（≥5 mm）；无论三种重组蛋白单一还是联合诱导MTB活菌致敏豚鼠，所产生的红晕/硬结在12~24 h的直径平均值均大于24~48 h，差异均具有统计学意义（P ＜0.0001）；此外，三种重组蛋白联合诱导在12~24 h产生的红晕/硬结的直径平均值大于单一蛋白，差异具有统计学意义（P=0.0005、P=0.0012、P=0.0149）；三种重组蛋白联合诱导24~48 h红晕/硬结的直径平均值大于单一蛋白，差异具有统计学意义（P=0.0014、P=0.0005、P=0.0067）。三种重组蛋白单一和联合所诱导的MTB灭活菌菌体或BCG致敏豚鼠的皮肤反应均为阴性，重组蛋白诱导MTB活菌、MTB灭活菌体和BCG致敏豚鼠的DTH数据有显著性差异（P值均＜0.0001）；而TB-PPD所诱导的不同结核分枝杆菌致敏豚鼠的DTH反应均为阳性。

结论：本研究成功获得纯化的rEC、Rv2654c、Rv2645重组蛋白，并对重组蛋白进行效力测定；重组蛋白单一和联合诱导MTB活菌致敏豚鼠的DTH反应在12~24 h所产生的红晕或硬结均较24~48 h更明显，且重组蛋白联合使用比任何一种蛋白单独使用的灵敏性更高；重组蛋白单一和联合使用均可鉴别MTB感染、MTB灭活菌体及BCG致敏豚鼠，重组蛋白与TB-PPD相比具有更好的特异性。

Background and objective: Tuberculosis (TB) is a chronic infectious disease caused by mycobacterium tuberculosis (MTB) infection. In recent years, with the prevalence of drug-resistant TB (DR-TB) and the increase of of MTB and HIV coinfection, particular, especially the lack of specific and sensitive diagnostic methods for latent tuberculosis and latent MTB infection (LTBI) further,  the prevention and control of TB becomes more and more difficult. How to achieve the early diagnosis, standard treatment and control of drug-resistant TB is still a hot and difficult issue in the global TB prevention and control research. Tuberculin skin test (TST) is the most widely used cellular immune diagnosis method at present, but its detection results have a high false positive rate, so it is urgent to find a new MTB specific antigen for TST detection. Culture filtrate protein (CFP10), early secreting antigen target 6 (ESAT6), Rv2654c and Rv2645 are specific proteins that have been discovered to be expressed in human pathogenic MTB strains in recent years, but they are not expressed in BCG strain. To date, the application value of the above dominant antigens in the diagnosis of TST is still unclear.

Materials and methods: The recombinant expression plasmids of esat6-cfp10 (rEC), Rv2654c and Rv2645 were constructed by genetic engineering technology, and the sequences of the recombinant plasmids were determined by restriction endonuclease digestion and direct sequencing. rEC, Rv2654c and Rv2645 recombinant expression plasmids were transformed into strain BL21 (DE3) of E. coli. The expression conditions of recombinant protein were optimized. The recombinant protein was purified by affinity Ni column chromatography, anion exchange CaptoQ chromatography and molecular sieve Superdex 200 chromatography, and verified by Western blot and mass spectrometry. TB-PPD was used as the reference to determine the efficacy of the recombinant protein. The DTH guinea pig model with live MTB, killed MTB and BCG sensitization was constructed. Tuberculin pure protein derivative (TB-PPD) was used as a positive control. Recombinant protein rEC, Rv2654c and Rv2645 were injected into three groups of guinea pig models by single or combined injection, and the horizontal and vertical diameters of local redness/induration were recorded at 12, 24 and 48 hours after injection, and the mean diameters at 12 to 24 hours and 24 to 48 hours were compared and analyzed to evaluate DTH intensity.

Results: After double enzyme digestion, agarose gel electrophoresis and DNA sequencing, the gene sequences of rEC, Rv2654c and Rv2645 in the pet-28a recombinant expression plasmids were consistent with the known sequences of target genes, demonstrated that the recombinant expression plasmids were successfully constructed. The recombinant plasmids were transformed into E. coli BL21 (DE3) and the optimized eapression conditions included growth until an absorbance of 0.6~0.8 (measured at 600 nm) with 0.1mmol /L IPTG and 30℃. SDS-PAGE gel electrophoresis showed that all recombinant proteins were expressed in the form of soluble proteins. High purity recombinant protein rEC, Rv2654c and Rv2645 (purity > 95%) were obtained by affinity Ni column chromatography, anion exchange chromatography and molecular sieve chromatography. Western Blot assay confirmed that the rEC, Rv2654c and Rv2645 proteins were consistent with the expected molecular weight of 26.5 kda, 6.5 kda and 16.7 kda, respectively. The protein spectrum analysis showed that the recombinant protein sequences were correct. The results of the determination of recombinant protein potency showed that the recombinant proteins of 5.0 μg/mL rEC, 10.0 μg/mL Rv2654c and 10.0 μg/mL Rv2645 were basically consistent with the results of the skin test of 1.0 μg/mL TB-PPD. The results of the immune function experiment showed that in the DTH test of guinea pigs sensitized by live MTB induced by recombinant protein, the average diameter of rEC（5.0 μg/mL）, Rv2654c（10.0 μg/mL）, Rv2645（10.0 μg/mL） and [rEC（3.3 μg/mL）+Rv2654c（3.3 μg/mL）+Rv2645（3.3 μg/mL）] were 12.3mm, 11.8mm, 12.3mm and 15.8mm, respectively, from 12 to 24 hours.The average diameters generated from 24 to 48 hours were 9.3mm, 8.8mm, 8.7 mm and 13.0mm, respectively. The results of the skin tests induced by recombinant proteins were all positive (≥5mm). No matter whether the three recombinant proteins were single or combined to induce live MTB sensitized guinea pigs, the average diameter of the resulting redness/indentation at 12-24 hours was greater than that at 24-48 hours, and the differences were statistically significant (P < 0.0001). In addition, the average diameter of the redness/indentation induced by the combination of the three recombinant proteins at 12-24 hours was greater than that of the single protein, and the differences were statistically significant (P=0.0005, P=0.0012, P=0.0149). In addition, the mean diameter of redness/indentation induced by the combination of the three recombinant proteins for 24 -48 hours was greater than that of the single protein, and the difference was statistically significant (P=0.0014, P=0.0005, P=0.0067). However, in the DTH test of guinea pigs sensitized by killed MTB or BCG, the skin reactions induce by single recombinant protein or combination of the three recombinant proteins were all negative. There were significant differences in the DTH data of recombinant protein-induced live MTB, killed MTB and BCG sensitized guinea pigs (all P values < 0.0001). While the DTH responses of guinea pigs sensitized by different mycobacterium tuberculosis induced by TB-PPD were all positive and there were no differences in the DTH data of TB-PPD -induced live MTB, killed MTB and BCG sensitized guinea pigs.

Conclusions: Our results showed that rEC, Rv2654c and Rv2645 recombinant proteins were successfully expressed and purified. The potency of these recombinant proteins was determined. The DTH response of guinea pigs sensitized by live MTB induced by single or combined use of three kinds of recombinant proteins was more obvious at 12~24 h than at 24~48 h. The combined use of recombinant proteins was more sensitive than that of any single protein. Single and combined use of recombinant proteins can identify live MTB, killed MTB and BCG sensitized guinea pigs. Compared with TB-PPD, recombinant proteins have better specificity.

缩略词表 1
第一章 引言 3
第二章 综述 6
2.1 结核病的流行及危害 6
2.2 结核分枝杆菌的病原学 8
2.2.1 结核分枝杆菌的形态结构及菌体成分 8
2.2.2 结核分枝杆菌的基因组结构 9
2.3 结核分枝杆菌的致病性和免疫性 10
2.3.1 结核分枝杆菌的致病性 10
2.3.2 结核分枝杆菌的易感人群 11
2.3.3 结核分枝杆菌的感染类型 12
2.3.4 结核分枝杆菌感染的结局 12
2.3.5 抗结核分枝杆菌的免疫机制 13
2.3.6 结核分枝杆菌的超敏反应 14
2.4 结核病的诊断方法 15
2.4.1 胸部X射线 16
2.4.2 细菌学诊断 17
2.4.3 免疫学诊断 18
2.5 结核分枝杆菌诊断优势抗原的研究进展 22
2.6 展望 24
第三章 材料与方法 26
3.1 实验材料 26
3.1.1 基因合成 26
3.1.2 菌株和质粒 26
3.1.3 实验动物 27
3.1.4 实验试剂 27
3.2 仪器设备 28
3.3 试剂配制 29
3.4 实验方法 31
3.4.1 重组质粒的构建及鉴定 31
3.4.2 目的蛋白的原核表达 33
3.4.3 重组蛋白的纯化和理化性质测定 36
3.4.4 重组蛋白效力评价及免疫功能研究 39
3.5 数据分析 42
第四章 结果 43
4.1 结核分枝杆菌重组蛋白的表达及纯化 43
4.1.1 重组质粒的鉴定 43
4.1.2 重组蛋白的诱导表达 45
4.1.3 重组蛋白的表达条件优化 45
4.1.4 重组蛋白的纯化 50
4.2 重组蛋白的鉴定 52
4.2.1 Western Blot检测 52
4.2.2 质谱分析 52
4.3 重组蛋白诱导结核分枝杆菌活菌致敏豚鼠的效力评价 54
4.4 重组蛋白免疫功能研究 56
4.4.1 重组蛋白对MTB活菌致敏豚鼠的DTH试验结果呈阳性 56
4.4.2 重组蛋白对MTB灭活菌体免疫豚鼠的DTH试验结果呈阴性 58
4.4.3 重组蛋白对BCG致敏豚鼠的DTH试验结果呈阴性 59
4.4.4 重组蛋白和TB-PPD诱导不同分枝杆菌免疫豚鼠的DTH结果比较 60
第五章 讨论 62
第六章 结论 65
参考文献 66
致谢 75
北京大学学位论文原创性声明和使用授权说明 76
学位论文答辩委员会名单 77
个人简历、在学期间发表的学术论文与研究成果 78

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