發(fā)布時(shí)間：2018-09-01 17:52

【摘要】： 研究背景: 弓形蟲(chóng)是一種專性細(xì)胞內(nèi)寄生的機(jī)會(huì)致病原蟲(chóng),生活史復(fù)雜,涉及的組織器官多,可引起人獸共患的弓形蟲(chóng)病。其危害嚴(yán)重程度在再現(xiàn)疾病之中僅次于結(jié)核;孕婦感染弓形蟲(chóng)可引起流產(chǎn)、早產(chǎn)、畸胎、死胎,嬰幼兒先天性弓形蟲(chóng)病,小兒智力障礙等,對(duì)人口素質(zhì)和優(yōu)生優(yōu)育和計(jì)劃生育有嚴(yán)重影響。近年來(lái),弓形蟲(chóng)腦炎已成為艾滋病患者的主要死亡原因之一;也是器官移植患者死亡和精神病發(fā)病的主要原因之一。危害如此嚴(yán)重,診斷、治療和預(yù)防卻還存在很多問(wèn)題亟需解決。治療尚無(wú)十分理想的藥物,采用的化學(xué)藥物有治療周期長(zhǎng),藥物毒副作用大,不能根治等缺陷,加上至今還無(wú)一種藥物能夠殺滅包囊內(nèi)的蟲(chóng)體,加之弓形蟲(chóng)的重復(fù)感染十分迅速,故雖經(jīng)多方面的努力,弓形蟲(chóng)病仍未得以很好地遏制。因此,研制有效的新藥和安全高效的弓形蟲(chóng)病疫苗,已成為人們的迫切要求,而廉價(jià)、安全、高效的疫苗無(wú)疑是最為經(jīng)濟(jì)、實(shí)用的防治措施。弓形蟲(chóng)病疫苗候選分子的篩選一直是研究中的瓶頸,也是研究的焦點(diǎn)和熱點(diǎn)。本工作在前期研究的基礎(chǔ)上進(jìn)一步篩選、鑒定更有效的疫苗候選基因或藥物分子靶標(biāo),制備核酸疫苗進(jìn)行動(dòng)物實(shí)驗(yàn)并闡明這個(gè)基因的功能、作用機(jī)制及其潛在的應(yīng)用價(jià)值。 研究目的: (1)通過(guò)制備具有保護(hù)性的單克隆抗體,篩選、鑒定弓形蟲(chóng)病疫苗的新的候選基因。 (2)構(gòu)建該基因的DNA疫苗,觀察其動(dòng)物保護(hù)效果以及對(duì)宿主機(jī)體免疫系統(tǒng)的影響。 (3)應(yīng)用RNA干擾技術(shù),使獲得的候選基因在轉(zhuǎn)錄后水平沉默,從而使其所表達(dá)的蛋白下降或者缺失,以期進(jìn)一步闡明該基因的功能和作用機(jī)制,對(duì)該基因的應(yīng)用前景進(jìn)行評(píng)估,并希望得到弓形蟲(chóng)的減毒蟲(chóng)株,用于制備減毒活疫苗。 研究方法: (1)應(yīng)用雜交瘤技術(shù)獲得抗弓形蟲(chóng)的單克隆抗體,體內(nèi)外實(shí)驗(yàn)觀察其保護(hù)性效果。以弓形蟲(chóng)單克隆抗體作為探針免疫篩選弓形蟲(chóng)速殖子cDNA文庫(kù),獲得其對(duì)應(yīng)的基因序列。經(jīng)Blast(http://www.ncbi.nlm.nih.gov/BLAST和(http://www.toxodb.org/ToxoDB.html.)進(jìn)行同源性比較,登錄GenBank并獲得新基因登錄序列號(hào)。 (2)采用雙色免疫熒光抗體定位和真核細(xì)胞內(nèi)表達(dá)等方法對(duì)該基因編碼的蛋白質(zhì)進(jìn)行鑒定,應(yīng)用生物信息學(xué)對(duì)基因編碼蛋白的B細(xì)胞表位、氨基酸序列、蛋白質(zhì)結(jié)構(gòu)等進(jìn)行預(yù)測(cè)和同源性分析。 (3)以攜帶wx2基因的pBluescript7C3-C3質(zhì)粒DNA為模板,通過(guò)PCR擴(kuò)增基因wx2的ORF;以攜帶wx基因的pBluescript2B9-G1質(zhì)粒DNA為模板,經(jīng)PCR擴(kuò)增獲得wx基因的ORF,構(gòu)建wx2和wx新基因的DNA疫苗并免疫昆明小鼠,觀察小鼠死亡時(shí)間和檢測(cè)實(shí)驗(yàn)動(dòng)物的淋巴細(xì)胞轉(zhuǎn)化率、脾細(xì)胞CD_4~*與CD_8~+淋巴細(xì)胞比值、IFN-γ、血清特異抗體等,以期闡明對(duì)機(jī)體免疫系統(tǒng)的影響。 (4)應(yīng)用RNA干擾技術(shù)構(gòu)建新基因wx2,wx逆病毒表達(dá)載體,與真核表達(dá)質(zhì)粒pcDNA3-wx2,pcDNA3-wx一起,經(jīng)脂質(zhì)體共轉(zhuǎn)染真核細(xì)胞觀察體外表達(dá)效果;經(jīng)電穿孔法導(dǎo)入弓形蟲(chóng)體內(nèi),獲得體內(nèi)能持續(xù)表達(dá)siRNA分子的可遺傳的RNA干擾蟲(chóng)株。采用Western-blotting,RT-PCR,Northern-blotting等方法鑒定其蛋白質(zhì)及mRNA水平變化效果;并對(duì)有基因沉默效果的干擾蟲(chóng)株進(jìn)行動(dòng)物感染實(shí)驗(yàn),觀察其的毒力變化;以研究減毒活疫苗的潛在價(jià)值。 研究結(jié)果: (1)獲得了兩株抗弓形蟲(chóng)的單克隆抗體7C3-C3,289-G1,體外保護(hù)性實(shí)驗(yàn)提示能抑制弓形蟲(chóng)對(duì)宿主細(xì)胞的侵襲以及在宿主細(xì)胞內(nèi)的繁殖,其細(xì)胞感染率分別為28%和32%,對(duì)照組為85.2%;50個(gè)HeLa細(xì)胞中納蟲(chóng)泡內(nèi)平均蟲(chóng)體數(shù)目為5.18±3.34與5.50±2.36,對(duì)照組為11.12±4.29。被動(dòng)轉(zhuǎn)移實(shí)驗(yàn)提示其能夠延長(zhǎng)RH株弓形蟲(chóng)攻擊小鼠的生存時(shí)間(7.2±0.42和7.0±1.41d,對(duì)照組為5.0d),表明這兩個(gè)單抗具有一定的抗弓形蟲(chóng)感染的保護(hù)力。 (2)通過(guò)免疫篩選弓形蟲(chóng)速殖子cDNA文庫(kù),獲得了兩個(gè)單抗所對(duì)應(yīng)的基因序列,經(jīng)同源性比對(duì)發(fā)現(xiàn)它們?yōu)樾禄?GenBank的登錄號(hào)為AY238892和AY208994。通過(guò)雙色免疫熒光定位和體外表達(dá)以及生物信息學(xué)分析對(duì)兩個(gè)新基因進(jìn)行了鑒定,證明WX2為一新的功能膜分子,分子量為49kDa;WX為弓形蟲(chóng)60S核糖體L7蛋白,分子量為47kDa,位于胞質(zhì)中。 (3)成功構(gòu)建了新基因wx2,wx的真核表達(dá)質(zhì)粒,作為DNA疫苗免疫動(dòng)物顯示pcDNA3-wx2與pcDNA3-wx能顯著延長(zhǎng)弓形蟲(chóng)RH株攻擊感染的生存時(shí)間,分別達(dá)289.14±46.81h和284.29±47.30h,對(duì)照組僅存活176.4±1.98h和172±1.88h,而且攻擊感染30天后疫苗接種組21只實(shí)驗(yàn)鼠中均有4只小鼠存活,顯示其潛在的應(yīng)用價(jià)值。對(duì)免疫鼠研究發(fā)現(xiàn),pcDNA3-wx2能刺激機(jī)體產(chǎn)生較高水平的IFN-γ(P＜0.05);pcDNA3-wx2與pcDNA3-wx都能使宿主脾細(xì)胞CD_4~+/CD_8~+比值下降,及產(chǎn)生特異性抗體,與對(duì)照組相比,P值＜0.05;但抗體效價(jià)的高低與保護(hù)力的強(qiáng)弱并不平行;提示該DNA疫苗主要以CD_8~+的CTL細(xì)胞途徑激發(fā)機(jī)體的抗弓形蟲(chóng)效應(yīng)。 (4)成功構(gòu)建了5個(gè)針對(duì)基因wx2,wx的干擾表達(dá)質(zhì)粒,獲得了2株部分沉默的RNA干擾蟲(chóng)株wx2b-i和wxB-i蟲(chóng)株。經(jīng)過(guò)體外表達(dá)以及RT-PCR,Northern-blotting等鑒定,證實(shí)基因wx2,wx mRNA水平及蛋白質(zhì)的表達(dá)均有部分下降。干擾蟲(chóng)株wx2b-i腹腔接種小鼠后其存活時(shí)間明顯延長(zhǎng),平均存活時(shí)間為235.4±15.4h,陰性組C-i和野生型RH株組分別為161.2±11.98h與165.4±6.09h,統(tǒng)計(jì)學(xué)分析具有顯著性差異,發(fā)病及死亡時(shí)間推遲,表明干擾株的毒力有所降低。 結(jié)論: (1)抗弓形蟲(chóng)的單克隆抗體7C3-C3,289-G1對(duì)于弓形蟲(chóng)的感染具有一定的保護(hù)性,能抑制弓形蟲(chóng)對(duì)宿主細(xì)胞的侵襲以及在宿主細(xì)胞內(nèi)的繁殖;被動(dòng)轉(zhuǎn)移實(shí)驗(yàn)提示能夠延長(zhǎng)RH株弓形蟲(chóng)攻擊小鼠的生存時(shí)間。 (2)WX2為一新的弓形蟲(chóng)功能膜分子,分子量為49kDa;WX為弓形蟲(chóng)60S核糖體L7蛋白,分子量為47kDa,定位于胞質(zhì)中。wx2,wx是兩個(gè)較好的弓形蟲(chóng)疫苗候選分子。 (3)成功建立了含新基因wx2,wx的DNA疫苗,能顯著延長(zhǎng)弓形蟲(chóng)強(qiáng)毒(RH株)攻擊感染動(dòng)物的生存時(shí)間,顯示其潛在應(yīng)用價(jià)值。對(duì)宿主免疫系統(tǒng)的影響觀察提示其主要以CD_8~+的CTL細(xì)胞途徑激發(fā)機(jī)體的抗弓形蟲(chóng)效應(yīng)。 (4)成功構(gòu)建了5個(gè)針對(duì)基因wx2,wx的干擾表達(dá)載體,并獲得了兩株部分沉默的RNA干擾蟲(chóng)株wx2b-i、wxB-i,其中wx2b-i蟲(chóng)株的毒力有所降低,為進(jìn)一步研究基因wx2,wx的功能與作用機(jī)制奠定了基礎(chǔ)。
[Abstract]:Research background:
Toxoplasma gondii is a specific intracellular parasitic opportunistic pathogenic protozoan with a complex life cycle, involving many tissues and organs, and can cause toxoplasmosis zoonosis. The severity of toxoplasmosis is second only to tuberculosis in the recurrence of the disease; Toxoplasma gondii infection in pregnant women can cause abortion, premature delivery, teratoxoplasmosis, stillbirth, congenital toxoplasmosis in infants and children's intelligence. In recent years, Toxoplasma gondii encephalitis has become one of the main causes of death in AIDS patients and one of the main causes of death and psychosis in organ transplant patients. There are no ideal drugs for the treatment of toxoplasmosis. The chemical drugs used have many defects, such as long treatment cycle, toxic side effects, and no drug can kill the worms in the cysts, and the repeated infection of Toxoplasma gondii is very rapid. Therefore, despite many efforts, toxoplasmosis has not been well controlled. The preparation of new effective drugs and safe and efficient vaccine against Toxoplasma gondii has become an urgent requirement of people, and inexpensive, safe and efficient vaccine is undoubtedly the most economical and practical preventive measures. One-step screening, identification of more effective vaccine candidate genes or drug molecular targets, preparation of nucleic acid vaccine for animal experiments and to clarify the function of this gene, the mechanism of action and its potential application value.
Research purposes:
(1) To screen and identify new candidate genes for toxoplasmosis vaccine by preparing protective monoclonal antibodies.
(2) To construct the DNA vaccine of the gene and observe its protective effect on the host immune system.
(3) RNA interference technique was used to silence the candidate gene at the post-transcriptional level so that the expressed protein could be decreased or deleted, so as to further elucidate the function and mechanism of the gene, evaluate the application prospect of the gene, and hope to obtain attenuated Toxoplasma gondii strain for the preparation of live attenuated vaccine.
Research methods:
(1) Monoclonal antibodies against Toxoplasma gondii were obtained by hybridoma technique and their protective effects were observed in vitro and in vivo. The corresponding gene sequences were obtained by screening the DNA Library of Toxoplasma gondii tachyzoites with monoclonal antibodies as probes. Blast (http://www.ncbi.nlm.nih.gov/BLAST) and (http://www.toxodb.org/ToxoDB.html.) Homology comparison, login GenBank and get the new gene accession sequence number.
(2) The protein encoded by the gene was identified by immunofluorescent antibody localization and eukaryotic cell expression. The B cell epitope, amino acid sequence and protein structure were predicted and analyzed by bioinformatics.
(3) The ORF of wx2 gene was amplified by PCR using pBluescript7C3-C3 plasmid DNA carrying wx2 gene as template; the ORF of Wx gene was obtained by PCR using pBluescript2B9-G1 plasmid DNA carrying Wx gene as template; the DNA vaccine of wx2 and Wx gene was constructed and immunized Kunming mice to observe the death time of mice and detect the lymphocyte of experimental animals. The transformation rate, the ratio of CD_4~* to CD_8~+ lymphocyte, IFN-gamma and serum specific antibody were used to elucidate the effect on the immune system.
(4) Using RNA interference technology to construct a new gene wx2, Wx retroviral expression vector, together with the eukaryotic expression plasmid pcDNA3-wx2, pcDNA3-wx, the eukaryotic cells were co-transfected by liposome to observe the effect of expression in vitro; Toxoplasma gondii was transfected by electroporation, and the inheritable RNA interference strain was obtained. Ng, RT-PCR, Northern-blotting and other methods were used to identify the effect of protein and mRNA level changes. Animal infection experiments were carried out to observe the virulence changes of interfering insect strains with gene silencing effect, and to study the potential value of live attenuated vaccine.
Research findings:
(1) Two monoclonal antibodies against Toxoplasma gondii, 7C3-C3,289-G1, were obtained. Protective experiments in vitro showed that the infection rate of the host cells was 28% and 32% respectively, and that of the control group was 85.2%. The average number of parasites in the Nanovesicles of 50 HeLa cells was 5.18 (+ 3.34) and 5.50 (+ 2.36), respectively. The survival time of mice attacked by RH strain of Toxoplasma gondii was prolonged (7.2 (+ 0.42) and 7.0 (+ 1.41) days, while that of control group was 5.0 days), indicating that the two McAbs had certain protective effect against Toxoplasma gondii infection.
(2) Two McAbs were cloned from the DNA Library of Toxoplasma gondii tachyzoites by immunoscreening and identified as new genes by homology comparison. GenBank's login numbers were AY238892 and AY208994. Two new genes were identified by immunofluorescence localization, in vitro expression and bioinformatics analysis. A novel functional membrane molecule with a molecular weight of 49 kDa and a WX ribosomal L7 protein of 60S of Toxoplasma gondii, with a molecular weight of 47 kDa, is located in the cytoplasm.
(3) Eukaryotic expression plasmids of new genes wx2 and Wx were successfully constructed. As DNA Vaccine Immunized animals, pcDNA3-wx2 and pcDNA3-wx significantly prolonged the survival time of RH strain of Toxoplasma gondii infected by attack infection, reaching 289.14, 46.81 h and 284.29, 47.30 h, respectively. The control group only survived 176.4, 1.98 h and 172, 1.88 h, and 21 mice in the vaccination group Four of the mice survived, indicating their potential application value. Studies on immunized mice showed that pcDNA3-wx2 could stimulate the production of higher levels of IFN-gamma (P The level of the DNA vaccine was not parallel to the protective power, suggesting that the anti-Toxoplasma gondii effect of the DNA vaccine was mainly stimulated by CD 8~+ CTL cell pathway.
(4) Five interfering expression plasmids were successfully constructed and two partially silenced RNA interfering strains wx2b-i and wxB-i were obtained. After in vitro expression, RT-PCR, Northern blotting and other identification, the expression of wx2, Wx mRNA and protein were partially decreased. The average survival time was 235.4 + 15.4 H. The virulence of C-i and wild type RH strains in negative group was 161.2 + 11.98 h and 165.4 + 6.09 h respectively. There was significant difference between the two groups. The time of onset and death was delayed, indicating that the virulence of the interfering strains was decreased.
Conclusion:
(1) Anti-Toxoplasma gondii monoclonal antibody 7C3-C3,289-G1 has protective effect on the infection of Toxoplasma gondii, and can inhibit the invasion of Toxoplasma gondii to the host cells and the reproduction of the host cells. Passive transfer experiments suggest that RH strain of Toxoplasma gondii can prolong the survival time of mice.
(2) WX2 is a new functional membrane molecule of Toxoplasma gondii with a molecular weight of 49 kDa, and WX is a 60S ribosomal L7 protein of Toxoplasma gondii with a molecular weight of 47 kDa, which is located in the cytoplasm.
(3) A DNA vaccine containing a novel gene wx2 and Wx was successfully established, which could significantly prolong the survival time of infected animals attacked by Toxoplasma gondii virulent (RH strain) and showed its potential application value.
(4) Five interference vectors targeting wx2 and Wx genes were constructed successfully, and two partially silenced RNA interference strains wx2b-i and wxB-i were obtained. The virulence of wx2b-i strain was decreased, which laid a foundation for further study on the function and mechanism of wx2 and Wx genes.
【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】：2007
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 邢瑩;豬附紅細(xì)胞體JL-2基因的原核表達(dá)及重組蛋白間接ELISA方法的建立[D];延邊大學(xué);2012年

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