【摘要】： 研究背景: 弓形蟲是一種專性細(xì)胞內(nèi)寄生的機(jī)會致病原蟲,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;孕婦感染弓形蟲可引起流產(chǎn)、早產(chǎn)、畸胎、死胎,嬰幼兒先天性弓形蟲病,小兒智力障礙等,對人口素質(zhì)和優(yōu)生優(yōu)育和計劃生育有嚴(yán)重影響。近年來,弓形蟲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)防卻還存在很多問題亟需解決。治療尚無十分理想的藥物,采用的化學(xué)藥物有治療周期長,藥物毒副作用大,不能根治等缺陷,加上至今還無一種藥物能夠殺滅包囊內(nèi)的蟲體,加之弓形蟲的重復(fù)感染十分迅速,故雖經(jīng)多方面的努力,弓形蟲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病疫苗,已成為人們的迫切要求,而廉價、安全、高效的疫苗無疑是最為經(jīng)濟(jì)、實用的防治措施。弓形蟲病疫苗候選分子的篩選一直是研究中的瓶頸,也是研究的焦點和熱點。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)行動物實驗并闡明這個基因的功能、作用機(jī)制及其潛在的應(yīng)用價值。 研究目的: (1)通過制備具有保護(hù)性的單克隆抗體,篩選、鑒定弓形蟲病疫苗的新的候選基因。 (2)構(gòu)建該基因的DNA疫苗,觀察其動物保護(hù)效果以及對宿主機(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,對該基因的應(yīng)用前景進(jìn)行評估,并希望得到弓形蟲的減毒蟲株,用于制備減毒活疫苗。 研究方法: (1)應(yīng)用雜交瘤技術(shù)獲得抗弓形蟲的單克隆抗體,體內(nèi)外實驗觀察其保護(hù)性效果。以弓形蟲單克隆抗體作為探針免疫篩選弓形蟲速殖子cDNA文庫,獲得其對應(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。 (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,觀察小鼠死亡時間和檢測實驗動物的淋巴細(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;并對有基因沉默效果的干擾蟲株進(jìn)行動物感染實驗,觀察其的毒力變化;以研究減毒活疫苗的潛在價值。 研究結(jié)果: (1)獲得了兩株抗弓形蟲的單克隆抗體7C3-C3,289-G1,體外保護(hù)性實驗提示能抑制弓形蟲對宿主細(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%,對照組為85.2%;50個HeLa細(xì)胞中納蟲泡內(nèi)平均蟲體數(shù)目為5.18±3.34與5.50±2.36,對照組為11.12±4.29。被動轉(zhuǎn)移實驗提示其能夠延長RH株弓形蟲攻擊小鼠的生存時間(7.2±0.42和7.0±1.41d,對照組為5.0d),表明這兩個單抗具有一定的抗弓形蟲感染的保護(hù)力。 (2)通過免疫篩選弓形蟲速殖子cDNA文庫,獲得了兩個單抗所對應(yīng)的基因序列,經(jīng)同源性比對發(fā)現(xiàn)它們?yōu)樾禄?GenBank的登錄號為AY238892和AY208994。通過雙色免疫熒光定位和體外表達(dá)以及生物信息學(xué)分析對兩個新基因進(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疫苗免疫動物顯示pcDNA3-wx2與pcDNA3-wx能顯著延長弓形蟲RH株攻擊感染的生存時間,分別達(dá)289.14±46.81h和284.29±47.30h,對照組僅存活176.4±1.98h和172±1.88h,而且攻擊感染30天后疫苗接種組21只實驗鼠中均有4只小鼠存活,顯示其潛在的應(yīng)用價值。對免疫鼠研究發(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,與對照組相比,P值＜0.05;但抗體效價的高低與保護(hù)力的強弱并不平行;提示該DNA疫苗主要以CD_8~+的CTL細(xì)胞途徑激發(fā)機(jī)體的抗弓形蟲效應(yīng)。 (4)成功構(gòu)建了5個針對基因wx2,wx的干擾表達(dá)質(zhì)粒,獲得了2株部分沉默的RNA干擾蟲株wx2b-i和wxB-i蟲株。經(jīng)過體外表達(dá)以及RT-PCR,Northern-blotting等鑒定,證實基因wx2,wx mRNA水平及蛋白質(zhì)的表達(dá)均有部分下降。干擾蟲株wx2b-i腹腔接種小鼠后其存活時間明顯延長,平均存活時間為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)計學(xué)分析具有顯著性差異,發(fā)病及死亡時間推遲,表明干擾株的毒力有所降低。 結(jié)論: (1)抗弓形蟲的單克隆抗體7C3-C3,289-G1對于弓形蟲的感染具有一定的保護(hù)性,能抑制弓形蟲對宿主細(xì)胞的侵襲以及在宿主細(xì)胞內(nèi)的繁殖;被動轉(zhuǎn)移實驗提示能夠延長RH株弓形蟲攻擊小鼠的生存時間。 (2)WX2為一新的弓形蟲功能膜分子,分子量為49kDa;WX為弓形蟲60S核糖體L7蛋白,分子量為47kDa,定位于胞質(zhì)中。wx2,wx是兩個較好的弓形蟲疫苗候選分子。 (3)成功建立了含新基因wx2,wx的DNA疫苗,能顯著延長弓形蟲強毒(RH株)攻擊感染動物的生存時間,顯示其潛在應(yīng)用價值。對宿主免疫系統(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)體的抗弓形蟲效應(yīng)。 (4)成功構(gòu)建了5個針對基因wx2,wx的干擾表達(dá)載體,并獲得了兩株部分沉默的RNA干擾蟲株wx2b-i、wxB-i,其中wx2b-i蟲株的毒力有所降低,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)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R392

【引證文獻(xiàn)】

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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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