本文選題：Cm + PCR��； 參考：《中南大學(xué)》2011年博士論文

【摘要】：泌尿生殖道沙眼衣原體(Chlamydia Trachomatis, CT)感染是細(xì)菌性性傳播疾病的主要原因�？股啬芡ㄟ^哺乳動(dòng)物細(xì)胞膜有效的治療CT感染,不過,由于缺乏明顯癥狀,許多感染者沒有尋求治療,從而導(dǎo)致上生殖道炎性并發(fā)癥,包括盆腔炎、異位妊娠、不孕不育等。顯然,最有效預(yù)防CT感染所致并發(fā)癥的方法是接種疫苗。雖然目前仍沒有獲得批準(zhǔn)的CT疫苗上市,然而50年前人類失敗的沙眼疫苗試驗(yàn)和此后大量的免疫學(xué)研究得出一個(gè)結(jié)論：采用亞單位疫苗防治CT感染和上生殖道并發(fā)癥是必要和可行的。然而,盡管在CT感染中,CT的胞內(nèi)復(fù)制及宿主對(duì)CT的抗原抗體反應(yīng)可顯著誘發(fā)炎性病理過程,但CT的確切發(fā)病機(jī)制仍不清楚,找到與CT致病密切相關(guān)的抗原分子是發(fā)展Ct亞單位疫苗的關(guān)鍵。 最近,大量的實(shí)驗(yàn)研究證實(shí)缺乏隱蔽性質(zhì)粒的鼠衣原體(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不引發(fā)任何已知的人類疾病,已被廣泛用于免疫生物學(xué)研究及CT疫苗抗原的尋找。迄今為止人們已成功應(yīng)用MoPn陰道感染小鼠模型,證明Th1主導(dǎo)的細(xì)胞免疫是防御泌尿生殖道衣原體感染所必須的；不攜帶質(zhì)粒的CT同樣表現(xiàn)出低致病性等。隱蔽質(zhì)粒不僅編碼它自己的8個(gè)開放閱讀框(ORFs),同時(shí)轉(zhuǎn)錄調(diào)節(jié)基因組22個(gè)開放閱讀框。所以,質(zhì)粒編碼和調(diào)節(jié)的開放閱讀框可能同時(shí)與衣原體致病性相關(guān)。 本研究采用MoPn泌尿生殖道感染模型,篩選可抵抗衣原體感染的保護(hù)性抗原。我們研究了7個(gè)受質(zhì)粒調(diào)節(jié)的抗原,克隆表達(dá)并純化蛋白經(jīng)肌肉注射免疫動(dòng)物,其中GlgP免疫誘導(dǎo)的保護(hù)性反應(yīng)最強(qiáng)烈。GlgP免疫不儀使小鼠衣原體感染后第14天陰道有機(jī)脫落物MoPn檢出率大為減少,還明顯減輕了上生殖道輸卵管積水的嚴(yán)重程度。GlgP誘導(dǎo)的保護(hù)作用與MoPn特異性抗體以及Thl主導(dǎo)T細(xì)胞反應(yīng)密切相關(guān)。這些觀察結(jié)果證明,可用肌內(nèi)注射純化蛋白免疫小鼠的方法,來(lái)鑒定CT疫苗的候選抗原,制備的疫苗可預(yù)防泌尿生殖道的CT感染和疾病。 第一部分鼠衣原體質(zhì)粒調(diào)控蛋白的克隆、原核表達(dá)及蛋白純化 目的：由于衣原體的隱蔽質(zhì)粒編碼22個(gè)調(diào)節(jié)蛋白,調(diào)節(jié)基因組編碼蛋白,因此研究被隱蔽質(zhì)粒編碼蛋白調(diào)節(jié)的蛋白,可以為CT亞單位疫苗的候選抗原篩選提供一定的實(shí)驗(yàn)依據(jù)。本研究旨在采用原核表達(dá)的方法獲得七種隱蔽質(zhì)粒調(diào)節(jié)蛋白,并采用親和層析的方法進(jìn)行純化。獲得純化蛋白為后續(xù)實(shí)驗(yàn)奠定基礎(chǔ)。 方法：本部分實(shí)驗(yàn)采用分子生物學(xué)的方法,從Genebank中獲取七種基因的基因序列,合成引物,以Cm標(biāo)準(zhǔn)株Nigg基因組DNA為模板,PCR獲得七種蛋白的基因序列,并將其克隆入原核表達(dá)載體pGEX-6p中。轉(zhuǎn)化大腸桿菌BL21,IPTG誘導(dǎo)表達(dá)。蛋白表達(dá)后利用GE公司的AKTA purifier系統(tǒng)進(jìn)行進(jìn)行純化,并采用Bradford的方法對(duì)蛋白進(jìn)行定量,以獲得足量的純化目的蛋白。 結(jié)果：經(jīng)測(cè)序驗(yàn)證,獲得七種序列完全正確的調(diào)控蛋白基因序列,并將其成功克隆入原核表達(dá)載體pGEX-6p中。IPTG誘導(dǎo)表達(dá)后,采用親和層析的方法進(jìn)行純化,獲得了電泳級(jí)純度的七種目的蛋白。 結(jié)論：成功表達(dá)純化出7個(gè)目的質(zhì)粒調(diào)控蛋白。 第二部分質(zhì)粒調(diào)控蛋白的疫苗保護(hù)性研究 目的：在第一部分中,我們通過原核表達(dá)以及親和層析獲得了七種質(zhì)粒調(diào)控蛋白的純化產(chǎn)物。因此在本部分的研究中,我們用陰道感染鼠衣原體的小鼠模型,來(lái)鑒定這七種抗原免疫注射小鼠后,其可否誘導(dǎo)抵抗鼠衣原體陰道感染的保護(hù)性免疫,以及哪種抗原誘導(dǎo)的保護(hù)性免疫最強(qiáng)烈。 方法：本部分實(shí)驗(yàn)采用鼠衣原體Nigg株(又名MoPn),采用常規(guī)方法進(jìn)行培養(yǎng)、純化、滴定,并感染Hela細(xì)胞獲得大量的鼠衣原體。采用肌肉注射的方法,將衣原體和抗原同時(shí)免疫小鼠,并采用CpG-IFA作為免疫佐劑。陰道感染后的4周內(nèi)每周用棉簽拭子收集陰道化驗(yàn)標(biāo)本,用以觀察陰道脫落上皮細(xì)胞中衣原體的含量情況。60天后處死小鼠分離生殖道組織,照相記錄結(jié)果并對(duì)輸卵管水腫程度進(jìn)行肉眼評(píng)分。 結(jié)果：糖原磷酸化酶(GlgP)免疫后明顯減少陰道內(nèi)感染后微生物的脫落。同時(shí),GlgP的小鼠免疫可明顯減輕衣原體感染引起輸卵管積水。 結(jié)論：糖原磷酸化酶免疫后明顯減少衣原體陰道內(nèi)感染量,減輕輸卵管積水程度。 第三部分GlgP蛋白疫苗的保護(hù)性機(jī)制研究 目的：在前兩個(gè)部分中,我們已經(jīng)獲得了七種純化的隱蔽質(zhì)粒調(diào)控蛋白,并且用這七種蛋白分別肌肉免疫小鼠,糖原磷酸化酶(GlgP)誘導(dǎo)的保護(hù)性免疫最強(qiáng)烈。因此,在本部分中,我們主要探討一下糖原磷酸化酶(GlgP)在體內(nèi)誘導(dǎo)保護(hù)性免疫的作用機(jī)制。 方法：ELISA檢測(cè)糖原磷酸化酶(GlgP)的免疫血清,鑒定體內(nèi)產(chǎn)生的抗體亞型；取各組免疫小鼠脾細(xì)胞,刺激后,測(cè)定脾細(xì)胞上清細(xì)胞因子水平；同時(shí),為進(jìn)一步分析GlgP的生物學(xué)特性及保護(hù)機(jī)制,我們采用Western-Blot技術(shù)對(duì)GlgP在衣原體的分布進(jìn)行分析。 結(jié)果：ELISA、免疫熒光實(shí)驗(yàn)以及不同細(xì)胞因子刺激免疫后的小鼠脾細(xì)胞實(shí)驗(yàn)均證實(shí)了Glgp免疫產(chǎn)生的抗體亞型主要是IgG2a,其保護(hù)機(jī)制是可誘導(dǎo)Thl型為主的細(xì)胞免疫應(yīng)答。GlgP分布與結(jié)構(gòu)蛋白MoMP一致,在衣原體EB和RB上均有分布,而在感染細(xì)胞的胞漿中沒有存在,GlgP在衣原體菌體高豐度的表達(dá)可能與其誘導(dǎo)的保護(hù)性免疫應(yīng)答有關(guān)。 結(jié)論：糖原合成酶誘導(dǎo)Thl型為主的細(xì)胞免疫應(yīng)答,應(yīng)用陰道感染模型可作為篩查衣原體疫苗的理想動(dòng)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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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本文編號(hào)：1737083