【作者】 李红；

【导师】 刘全忠；

【作者基本信息】 天津医科大学 ， 皮肤性病学， 2004， 硕士

【摘要】 衣原体是专性细胞内寄生的原核微生物，革兰氏染色阴性，属细菌范畴。衣原体引起的疾病相当广泛：在发达国家衣原体是性传播疾病的首位病原体，由它引起的非淋菌性尿道炎的发病率超过淋病，在性传播疾病中己跃居首位；沙眼衣原体(C．t)感染眼部引起的沙眼曾肆虐全世界，至今仍是全球可预防性眼盲的首要原因；衣原体感染动物可导致牛、羊流产，母鸡不能正常产卵，对养殖畜牧业造成很大危害；因此衣原体越来越成为人们关注的重要病原体。人们急切地期待衣原体疫苗的出现以控制衣原体的感染及蔓延，科技工作者在此领域已探索50余年，曾试验过多种疫苗，如：灭活或减毒的衣原体疫苗、亚单位疫苗、树突状细胞疫苗等，但迄今尚无成熟疫苗问世。近年来DNA疫苗研究的快速发展给衣原体疫苗研制带来新的希望，随着分子生物学技术突飞猛进的发展，基因工程技术日臻完善，衣原体的生物构成及基因序列日益明朗，衣原体的DNA疫苗很有发展潜力。 DNA疫苗是运用基因工程技术，将编码某种蛋白的外源基因与细菌质粒构建的重组体直接免疫机体，转染宿主细胞，使其表达保护性抗原，从而诱导机体产生特异性免疫的疫苗。 本实验的目的是构建C．t主要外膜蛋白(MOMP)编码基因ompA与pcDNA3质粒的重组体ompA-pcDNA3，即C．t的DNA疫苗，希望能为C．tDNA疫苗的动物实验研究提供材料。 实验中选用的真核表达载体为pcDNA3质粒，目的基因为C．t的MOMP编码基因ompA，使用的C．t标本来源于性传播疾病门诊患者的尿道及宫颈分泌物。标本经裂解处理后做为PCR扩增的模板，先用C．t种特异性引物扩增ompA基因，以此来筛选C．t阳性的临床标本；PCR产物经基因测序，确定C．t临床标本的血清型；然后用带限制性酶切位点的血清型特异性引物，PCR扩增此特定血清型的ompA基因；扩增的目的基因与载体经酶切后，连接重组，转化大肠杆菌；最后重组子经抗生素平板筛选、PCR扩增筛选、酶切鉴定后，阳性重组子送测序公司，进行重组子的DNA序列测定。 实验显示：PCR扩增方法筛选C．t阳性的临床标本，扩增产物经琼脂糖凝胶电泳，扩增产物大小约1.2Kb，基因测序确定是C．t J血清型；PCR扩增天津医科大学硕士研究生论文J型c.t MoMP编码基因。mPA，扩增产物经琼脂糖凝胶电泳，扩增产物片段约1.OKb，与预期的1022bp一致;PCR扩增方法筛选重组子ompA一PcDNA3，琼脂糖凝胶电泳提示。mpA基因存在于PcDNA3质粒中;双酶切法筛选重组子，酶切产生了5.4Kb和1 .oKb两个片段，进一步证明omPA基因存在于PcDNA3质粒中;阳性重组子进行基因测序，证明插入PcDNA3载体中的墓因片段与genebar水中J型C .t的 omPA基因序列一致。上述结果证明本实验成功构建了C.tJ型野生株。mpA基因的重组子ompA一peDNA3。更多还原

【Abstract】 Chlamydia is an obligate , intracellular, Gram negative bacterial pathogen. The diseases evoked by chlamydia are quite wide. In developed countries, the pathogen is the leading cause of sexually transmitted disense. Infection in the eyes results in trachoma which once spreaded widely in the whole world. By now trachoma is still the leading cause of preventable blindness in the world. Chlamydia infection can also make cattle and sheep abort, hens not lay eggs normally. So chlamydia has made huge damage to both mankind and anminals. Chlamydia has became the important pathogen which absorbs people’s attention. Chlamydial vaccion has the probability of controling chlamydial infection and spread. People have studied chlamydia vaccine for more then 50 years. Many kinds of vaccine have been tested, such as the inactivated pathogen vaccine, the live attenuated pathogen vaccine, the subunit vaccine, the dendritic cell vaccine and so on. But there isa’t a mature vaccine that can be used now. In recent years, the fast and deep research OB the DNA vaccine brings new hope for stydying the chlamydia DNA vaccine.The DNA vaccine, which is a recombinant of plasmid with a gene fagment encoding antigen protein, vaccinate the organism directly , transform the organism cells, express the protective antigen, and then induce the organism to obtain specefic immunity.Our test is aming at constructing an recombinant of ompA gene from the chlamydia trachomatis wild strain. We choose pcDNA3 plasmid as the vector and ompA gene which encodes chlamydia trachomatis (C.t) major outer membrane protein as the target DNA. Clinical chlamydia trachomatis samples,urethral and cervical secreations, were collected from persons who presented to the outpatient department of the sexually transmitted disease. After lysised, clinical samples wert used as template for polymerase chain reaction(PCR). Firstly ,to select C.t positivesamples, we amplified ompA gene through PCR using C.t species specific primier, and then we ascertain the C.t clinical specimen’s serotype by the DNA sequence method. Secondly, through polymerase chain reaction, we amplified specific serotype C.t ompA gene using serotype specific primier. Then we enzymatically inserted DNA fragment into plasmid vector, we used three methods to screen positive recombinant, including culturing the E.coli on LB agarose plate containing ampicillin, amplifing ompA gene from recomibant through the PCR method, and digesting recombinant by restriction endonuclease. Finally, we sequenced the inserted DNA fragment by sending the positive recombinant to the sequence corporation.The results are as follows. By using the PCR method to screen positive C.t clinical samples, the obtained DNA fragment is about 1.2Kb long. The sample which was sended to be sequenced was proved to be C.t J serotype. Agarose gel electrophoresis manifests that the amplified C.t J serotype ompA gene is a little longer than 1.0Kb. The length of the amplified DNA fragment is consistent whih what we have anticipated. When positive recombinant is screened by the PCR method, agarose gel electrophoresis manifests that ompA gene lies in pcDNA3 plasmid. Digested recombinant with restriction endonuclease, we obtained two DNA fragments of separately 5.4Kb and 1.0Kb. The result also confirms that the ompA gene lies in pcDNA3 plasmid. When we sequenced the inserted DNA fragment, the result proved that the inserted gene has the same sequence with C.t J serotype ompA gene provided by genebank. Above results manifest that our test have successfully construsted an recombinant of ompA gene from chlamydia trachomatis wild strain J serotype.更多还原