糖原磷酸化酶对鼠衣原体泌尿生殖道感染的保护性研究

发布时间：2018-04-11 18:03

本文选题：Cm + PCR　；参考：《中南大学》2011年博士论文

【摘要】：泌尿生殖道沙眼衣原体(Chlamydia Trachomatis, CT)感染是细菌性性传播疾病的主要原因。抗生素能通过哺乳动物细胞膜有效的治疗CT感染,不过,由于缺乏明显症状,许多感染者没有寻求治疗,从而导致上生殖道炎性并发症,包括盆腔炎、异位妊娠、不孕不育等。显然,最有效预防CT感染所致并发症的方法是接种疫苗。虽然目前仍没有获得批准的CT疫苗上市,然而50年前人类失败的沙眼疫苗试验和此后大量的免疫学研究得出一个结论：采用亚单位疫苗防治CT感染和上生殖道并发症是必要和可行的。然而,尽管在CT感染中,CT的胞内复制及宿主对CT的抗原抗体反应可显著诱发炎性病理过程,但CT的确切发病机制仍不清楚,找到与CT致病密切相关的抗原分子是发展Ct亚单位疫苗的关键。最近,大量的实验研究证实缺乏隐蔽性质粒的鼠衣原体(Chlamydia muridarum,又名MoPn)失去了致小鼠上生殖道感染的能力,而野生型的MoPn仍具有高致病性。由于MoPn不引发任何已知的人类疾病,已被广泛用于免疫生物学研究及CT疫苗抗原的寻找。迄今为止人们已成功应用MoPn阴道感染小鼠模型,证明Th1主导的细胞免疫是防御泌尿生殖道衣原体感染所必须的；不携带质粒的CT同样表现出低致病性等。隐蔽质粒不仅编码它自己的8个开放阅读框(ORFs),同时转录调节基因组22个开放阅读框。所以,质粒编码和调节的开放阅读框可能同时与衣原体致病性相关。本研究采用MoPn泌尿生殖道感染模型,筛选可抵抗衣原体感染的保护性抗原。我们研究了7个受质粒调节的抗原,克隆表达并纯化蛋白经肌肉注射免疫动物,其中GlgP免疫诱导的保护性反应最强烈。GlgP免疫不仪使小鼠衣原体感染后第14天阴道有机脱落物MoPn检出率大为减少,还明显减轻了上生殖道输卵管积水的严重程度。GlgP诱导的保护作用与MoPn特异性抗体以及Thl主导T细胞反应密切相关。这些观察结果证明,可用肌内注射纯化蛋白免疫小鼠的方法,来鉴定CT疫苗的候选抗原,制备的疫苗可预防泌尿生殖道的CT感染和疾病。第一部分鼠衣原体质粒调控蛋白的克隆、原核表达及蛋白纯化目的：由于衣原体的隐蔽质粒编码22个调节蛋白,调节基因组编码蛋白,因此研究被隐蔽质粒编码蛋白调节的蛋白,可以为CT亚单位疫苗的候选抗原筛选提供一定的实验依据。本研究旨在采用原核表达的方法获得七种隐蔽质粒调节蛋白,并采用亲和层析的方法进行纯化。获得纯化蛋白为后续实验奠定基础。方法：本部分实验采用分子生物学的方法,从Genebank中获取七种基因的基因序列,合成引物,以Cm标准株Nigg基因组DNA为模板,PCR获得七种蛋白的基因序列,并将其克隆入原核表达载体pGEX-6p中。转化大肠杆菌BL21,IPTG诱导表达。蛋白表达后利用GE公司的AKTA purifier系统进行进行纯化,并采用Bradford的方法对蛋白进行定量,以获得足量的纯化目的蛋白。结果：经测序验证,获得七种序列完全正确的调控蛋白基因序列,并将其成功克隆入原核表达载体pGEX-6p中。IPTG诱导表达后,采用亲和层析的方法进行纯化,获得了电泳级纯度的七种目的蛋白。结论：成功表达纯化出7个目的质粒调控蛋白。第二部分质粒调控蛋白的疫苗保护性研究目的：在第一部分中,我们通过原核表达以及亲和层析获得了七种质粒调控蛋白的纯化产物。因此在本部分的研究中,我们用阴道感染鼠衣原体的小鼠模型,来鉴定这七种抗原免疫注射小鼠后,其可否诱导抵抗鼠衣原体阴道感染的保护性免疫,以及哪种抗原诱导的保护性免疫最强烈。方法：本部分实验采用鼠衣原体Nigg株(又名MoPn),采用常规方法进行培养、纯化、滴定,并感染Hela细胞获得大量的鼠衣原体。采用肌肉注射的方法,将衣原体和抗原同时免疫小鼠,并采用CpG-IFA作为免疫佐剂。阴道感染后的4周内每周用棉签拭子收集阴道化验标本,用以观察阴道脱落上皮细胞中衣原体的含量情况。60天后处死小鼠分离生殖道组织,照相记录结果并对输卵管水肿程度进行肉眼评分。结果：糖原磷酸化酶(GlgP)免疫后明显减少阴道内感染后微生物的脱落。同时,GlgP的小鼠免疫可明显减轻衣原体感染引起输卵管积水。结论：糖原磷酸化酶免疫后明显减少衣原体阴道内感染量,减轻输卵管积水程度。第三部分GlgP蛋白疫苗的保护性机制研究目的：在前两个部分中,我们已经获得了七种纯化的隐蔽质粒调控蛋白,并且用这七种蛋白分别肌肉免疫小鼠,糖原磷酸化酶(GlgP)诱导的保护性免疫最强烈。因此,在本部分中,我们主要探讨一下糖原磷酸化酶(GlgP)在体内诱导保护性免疫的作用机制。方法：ELISA检测糖原磷酸化酶(GlgP)的免疫血清,鉴定体内产生的抗体亚型；取各组免疫小鼠脾细胞,刺激后,测定脾细胞上清细胞因子水平；同时,为进一步分析GlgP的生物学特性及保护机制,我们采用Western-Blot技术对GlgP在衣原体的分布进行分析。结果：ELISA、免疫荧光实验以及不同细胞因子刺激免疫后的小鼠脾细胞实验均证实了Glgp免疫产生的抗体亚型主要是IgG2a,其保护机制是可诱导Thl型为主的细胞免疫应答。GlgP分布与结构蛋白MoMP一致,在衣原体EB和RB上均有分布,而在感染细胞的胞浆中没有存在,GlgP在衣原体菌体高丰度的表达可能与其诱导的保护性免疫应答有关。结论：糖原合成酶诱导Thl型为主的细胞免疫应答,应用阴道感染模型可作为筛查衣原体疫苗的理想动物模型。
[Abstract]:Urogenital Chlamydia trachomatis (Chlamydia Trachomatis, CT) infection is a major cause of bacterial sexually transmitted diseases. Antibiotics can be infected by mammalian cell membrane, effective treatment of CT. However, due to the lack of obvious symptoms, many infected people do not seek treatment, resulting in reproductive tract inflammatory complications, including pelvic inflammatory disease, ectopic pregnancy. Infertility. Obviously, the most effective method of preventing complications caused by CT infection is vaccination. Although there is still no vaccine approved by the CT to get listed, but 50 years ago human failure trachoma vaccine test and then a large number of immunological studies to draw a conclusion: the subunit vaccine to prevent CT infection and reproductive tract complications it is necessary and feasible. However, in spite of CT infection, the antigen antibody reaction of CT intracellular replication and host of CT can significantly induce the inflammatory pathological process, but CT did The pathogenesis is still unclear, and it is the key to develop the Ct subunit vaccine to find the antigen molecules closely related to the pathogenesis of CT.
Recently, a large number of experimental studies confirmed the lack of cryptic plasmid of Chlamydia mouse (Chlamydia muridarum, aka MoPn) lost the ability of mice induced by upper genital tract infection, while the wild type MoPn has high pathogenicity. Because the MoPn does not cause any known human disease, has been widely used for biological studies and CT vaccine immunity antigen. So far have been successfully applied MoPn vaginal infection mouse model that Th1 cell immunity is necessary for leading defense of urogenital Chlamydia infection; not carrying plasmid CT also showed low pathogenicity. Plasmids encoding 8 not only its own open reading frame (ORFs), and transcription regulation the genome of 22 open reading frames. Therefore, plasmid encoding and regulating the open reading frame may also with Chlamydia pathogenicity.
This study uses MoPn urogenital tract infection model, screening resistant protective antigen of Chlamydia infection. We studied 7 by regulating plasmid antigen, cloning expression and purification of proteins by intramuscular injection of immune animal, the protective response induced by vaccination with GlgP.GlgP not only make the strongest immune mice fourteenth days after vaginal Chlamydia infection organic matter loss detection rate of MoPn is greatly reduced, but also significantly reduce the reproductive tract of hydrosalpinx severity of.GlgP induced protective effect with MoPn specific antibody and Thl dominant T cell responses are closely related. The observation results prove that the method can be used for intramuscular injection of purified mouse immune protein, to identify candidate antigen CT the vaccine, the vaccine preparation can prevent urogenital CT infection and disease.
Cloning, prokaryotic expression and protein purification of plasmids regulatory protein of Chlamydia
Objective: because of the 22 plasmids encoding Chlamydia proteins, regulating genome encoding protein, therefore research on concealed plasmid encoding protein regulating protein, can be a candidate antigen for CT subunit vaccine can provide some experimental basis for screening. The purpose of this study is to obtain seven kinds of cryptic plasmid regulated protein by prokaryotic expression, and the use of affinity chromatography. The purified protein was purified to lay the foundation for subsequent experiments.
Methods: this experiment using molecular biology, obtaining gene sequences of seven genes from the Genebank primers and Cm standard strain Nigg genomic DNA as template, PCR gene sequences of seven proteins, and then cloned into prokaryotic expression vector pGEX-6p was transformed into E.coli. BL21, induced by IPTG the expression of protein expression. After using the AKTA purifier system of GE company was purified, and uses the Bradford method to quantify protein, purify the protein to obtain enough.
Results: after sequencing, seven sequences of the seven regulatory proteins were successfully cloned into the prokaryotic expression vector pGEX-6p, and.IPTG was induced to express. After purification by affinity chromatography, we obtained the target protein of electrophoresis grade purity.
Conclusion: 7 target plasmids were successfully expressed and purified.
Study on vaccine protection of plasmid regulated protein in second parts
Objective: in the first part, we through prokaryotic expression and affinity chromatography purified product of seven kinds of plasmid regulatory proteins. Therefore, in the present study, we used a mouse model of Chlamydia trachomatis induced vaginal infection, to identify the seven immunization antigen in mice. It can induce protective immunity against Chlamydia trachomatis induced vaginal infection, and the protective immunity induced by antigen which most strongly.
Methods: the experiments using rat strains of Chlamydia pneumoniae Nigg (aka MoPn), using conventional methods of cultivation, purification, titration, and access to a large number of infected Hela cells. Methods the Chlamydia trachomatis induced by intramuscular injection, the chlamydia and antigen immunized mice, and using CpG-IFA as adjuvant. 4 weeks after vaginal infection with a cotton swab to collect vaginal swabs, the mice were sacrificed to observe tissue isolated from genitourinary tract Chlamydia vaginal exfoliated epithelial cells in content of.60 days, photographic recording and the results of tubal flesh eye edema degree score.
Results: glycogen phosphorylase (GlgP) immunization significantly reduced the microbial abscission after vaginal infection. Meanwhile, GlgP mice immunization could significantly reduce Chlamydia infection causing hydrosalpinx.
Conclusion: after immunization of glycogen phosphorylase, the infection of Chlamydia vagina can be reduced and the degree of hydrosalpinx is alleviated.
Study on the protective mechanism of the third part of GlgP protein vaccine
Objective: in the two part, we have obtained seven purified plasmids regulatory protein, and these seven proteins were immunized mice muscle glycogen phosphorylase (GlgP), protective immunity induced by the strongest. Therefore, in this part, we mainly discuss the glycogen phosphorylase (GlgP) in vivo. Induction of protective immunity.
Methods: ELISA assay of glycogen phosphorylase (GlgP) serum, identification of the body produces the antibody subtype; immune spleen cells were taken, stimulation, determination of spleen cells supernatant cytokine levels; at the same time, to further analyze the biological characteristics and the protective mechanism of GlgP, we adopt Western-Blot technology analysis of GlgP in the distribution of Chlamydia.
Results: ELISA, immunofluorescence experiments and different cytokines of spleen cells of mice after immunization experiments confirmed the antibody subtype Glgp immunity is mainly IgG2a, the protective mechanism is the cellular immune response induced by.GlgP Thl type distribution and structural protein MoMP, EB and RB were distributed in Chlamydia, and there is no infection in cell cytoplasm. The protective immune response induced by GlgP may be related to its expression in high abundance of Chlamydia bacteria.
Conclusion: the glycogen synthase induces the cellular immune response of the Thl type, and the application of the vaginal infection model can be used as an ideal animal model for screening Chlamydia vaccine.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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