鼠疫亞單位疫苗和口服活載體疫苗的研究
發(fā)布時(shí)間：2018-11-03 18:40
【摘要】： 鼠疫是由鼠疫耶爾森菌(Y.pestis)引起的人畜共患烈性傳染病,人間鼠疫包括肺鼠疫和腺鼠疫,腺鼠疫是通過跳蚤叮咬感染,Y.pestis遷移到肺部就會(huì)引起肺鼠疫感染,肺鼠疫能夠通過氣溶膠在人與人之間傳播,在不予治療的情況下死亡率高達(dá)100%。目前鼠疫仍是一個(gè)重要的公共衛(wèi)生問題,世界上每年還有上千感染病例,主要分布在非洲、亞洲和南美洲。由于傳統(tǒng)的鼠疫疫苗存在對(duì)肺鼠疫無效、副反應(yīng)強(qiáng)烈等缺陷,至今世界還沒有一種值得推廣的鼠疫疫苗。一種理想的鼠疫疫苗應(yīng)具備成本低、熱穩(wěn)定、能通過黏膜簡(jiǎn)便方法接種、能同時(shí)刺激機(jī)體產(chǎn)生體液和細(xì)胞免疫應(yīng)答等特點(diǎn),基于肺鼠疫的感染特點(diǎn),激發(fā)黏膜免疫反應(yīng)是預(yù)防肺鼠疫的研究重點(diǎn)。Y.pestis能產(chǎn)生很多由染色體或質(zhì)粒編碼的毒力因子,包括pgm、pst、Yops、F1抗原及V抗原等,由于這些毒力因子的致病作用機(jī)制尚不清楚,很難確定候選疫苗的組分。然而,諸多研究發(fā)現(xiàn),F1和V抗原是鼠疫兩大主要保護(hù)性抗原,也是鼠疫新型疫苗研究的重點(diǎn)。 本研究就是以F1和V抗原為材料,進(jìn)行鼠疫新型疫苗的探索,主要內(nèi)容包括: 1.從Y.pestis EV76株的質(zhì)粒中釣取編碼F1和V抗原的基因片段,并將二者融合克隆到原核表達(dá)載體pET11c上,轉(zhuǎn)入宿主菌E. coli BL21,用IPTG誘導(dǎo)表達(dá)重組融合蛋白rF1-V,純化后加入氫氧化鋁佐劑制備鼠疫候選亞單位疫苗。 2.將編碼F1-V融合蛋白的基因,克隆到原核表達(dá)載體asd-pTrc99A,轉(zhuǎn)入asd-大腸桿菌X6097,用電擊轉(zhuǎn)化經(jīng)減毒沙門氏菌中間宿主asd- X3730轉(zhuǎn)入減毒沙門氏菌終末宿主asd- X4072得到重組菌X4072(F1-V/99A),將菌體裂解液做Western-blot鑒定表達(dá)的F1-V融合蛋白。從而構(gòu)建出穩(wěn)定表達(dá)F1-V融合抗原的減毒沙門氏菌X4072(F1-V/99A),作為鼠疫口服疫苗候選株。 3.以小鼠為動(dòng)物模型進(jìn)行鼠疫候選DNA疫苗(裸質(zhì)粒F1-V/pVAX1)的安全性檢測(cè),通過接種動(dòng)物的大體觀察和稱重等方法,進(jìn)行急性毒性和長(zhǎng)期毒性試驗(yàn);提取組織DNA,通過PCR方法,檢測(cè)質(zhì)粒F1-V/pVAX1在小鼠組織的分布和滯留時(shí)間;用ELISA和ELISPOT方法進(jìn)行抗核抗體檢測(cè)。初步驗(yàn)證裸質(zhì)粒F1-V/pVAX1的安全性,為下一步口服活載體DNA疫苗的研究提供依據(jù)。 4.將編碼F1-V融合抗原的基因克隆到真核表達(dá)載體asd-pVAX1,轉(zhuǎn)入asd-大腸桿菌X6212,再依次將重組質(zhì)粒轉(zhuǎn)化asd-減毒沙門氏菌X3730、X4550,得到重組減毒沙門氏菌X4550(F1-V/AP),提取重組質(zhì)粒轉(zhuǎn)染COS-7細(xì)胞,用免疫組化和Western-blot
[Abstract]:Yersinia pestis is a zoonotic infectious disease caused by Yersinia pestis (Y.pestis). Human plague includes pneumonic plague and glandular plague, which is infected by flea bite, and the migration of Y.pestis to the lungs can cause pneumonic plague infection. Pneumonic plague can spread from person to person through aerosol, with a mortality rate of up to 100 kgs without treatment. Plague is still an important public health problem, with thousands of infections worldwide each year, mainly in Africa, Asia and South America. Because the traditional Yersinia pestis vaccine is ineffective to the pneumonic plague and has strong side effects, so far there is not a Yersinia pestis vaccine worth popularizing in the world. An ideal Yersinia pestis vaccine should have the characteristics of low cost, stable heat, simple method of inoculation through mucous membrane, stimulation of humoral and cellular immune response, etc., based on the characteristics of pneumonic plague infection. Y.pestis can produce many virulence factors encoded by chromosomes or plasmids, including pgm,pst,Yops,F1 antigen and V antigen. Because the pathogenicity of these virulence factors is unclear, it is difficult to determine the components of the candidate vaccine. However, many studies have found that F1 and V antigens are the two major protective antigens of plague, and they are also the focus of the study on the new vaccine of Yersinia pestis. In this study, F1 and V antigens were used as materials to explore a new vaccine for plague. The main contents were as follows: 1. The gene fragment encoding F1 and V antigen was isolated from the plasmid of Y.pestis EV76 strain and cloned into prokaryotic expression vector pET11c. The recombinant fusion protein rF1-V, was expressed by IPTG induced by E. coli BL21,. After purification, aluminum hydroxide adjuvant was added to prepare candidate subunit vaccine of Yersinia pestis. 2. The gene encoding F1-V fusion protein was cloned into prokaryotic expression vector asd-pTrc99A, into asd- Escherichia coli X6097. Asd- X3730, an intermediate host of attenuated Salmonella, was transformed into asd- X4072, the final host of attenuated Salmonella. The recombinant strain X4072 (F1-V/99A) was obtained by electroporation. The recombinant strain X4072 (F1-V/99A) was used as the F1-V fusion protein identified by Western-blot. Thus, attenuated Salmonella X4072 (F1-V/99A), which stably expressed F1-V fusion antigen, was constructed as an oral vaccine candidate for Yersinia pestis. 3. The safety of candidate DNA vaccine (naked plasmid F1-V/pVAX1) of Yersinia pestis was detected in mice as animal model. Acute and long-term toxicity tests were carried out by means of gross observation and weighing of inoculated animals. The distribution and retention time of plasmid F1-V/pVAX1 in mouse tissues were detected by PCR, and the antinuclear antibodies were detected by ELISA and ELISPOT methods. The safety of naked plasmid F1-V/pVAX1 was preliminarily verified, which provided the basis for the further study of oral live vector DNA vaccine. 4. The gene encoding F1-V fusion antigen was cloned into eukaryotic expression vector asd-pVAX1, into asd- Escherichia coli X6212, then transformed into asd- attenuated Salmonella X3730 X4550 in turn to obtain recombinant attenuated Salmonella X4550 (F1-V/AP). Recombinant plasmid was extracted and transfected into COS-7 cells. Immunohistochemistry and Western-blot were used.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392.1
本文編號(hào)：2308621
[Abstract]:Yersinia pestis is a zoonotic infectious disease caused by Yersinia pestis (Y.pestis). Human plague includes pneumonic plague and glandular plague, which is infected by flea bite, and the migration of Y.pestis to the lungs can cause pneumonic plague infection. Pneumonic plague can spread from person to person through aerosol, with a mortality rate of up to 100 kgs without treatment. Plague is still an important public health problem, with thousands of infections worldwide each year, mainly in Africa, Asia and South America. Because the traditional Yersinia pestis vaccine is ineffective to the pneumonic plague and has strong side effects, so far there is not a Yersinia pestis vaccine worth popularizing in the world. An ideal Yersinia pestis vaccine should have the characteristics of low cost, stable heat, simple method of inoculation through mucous membrane, stimulation of humoral and cellular immune response, etc., based on the characteristics of pneumonic plague infection. Y.pestis can produce many virulence factors encoded by chromosomes or plasmids, including pgm,pst,Yops,F1 antigen and V antigen. Because the pathogenicity of these virulence factors is unclear, it is difficult to determine the components of the candidate vaccine. However, many studies have found that F1 and V antigens are the two major protective antigens of plague, and they are also the focus of the study on the new vaccine of Yersinia pestis. In this study, F1 and V antigens were used as materials to explore a new vaccine for plague. The main contents were as follows: 1. The gene fragment encoding F1 and V antigen was isolated from the plasmid of Y.pestis EV76 strain and cloned into prokaryotic expression vector pET11c. The recombinant fusion protein rF1-V, was expressed by IPTG induced by E. coli BL21,. After purification, aluminum hydroxide adjuvant was added to prepare candidate subunit vaccine of Yersinia pestis. 2. The gene encoding F1-V fusion protein was cloned into prokaryotic expression vector asd-pTrc99A, into asd- Escherichia coli X6097. Asd- X3730, an intermediate host of attenuated Salmonella, was transformed into asd- X4072, the final host of attenuated Salmonella. The recombinant strain X4072 (F1-V/99A) was obtained by electroporation. The recombinant strain X4072 (F1-V/99A) was used as the F1-V fusion protein identified by Western-blot. Thus, attenuated Salmonella X4072 (F1-V/99A), which stably expressed F1-V fusion antigen, was constructed as an oral vaccine candidate for Yersinia pestis. 3. The safety of candidate DNA vaccine (naked plasmid F1-V/pVAX1) of Yersinia pestis was detected in mice as animal model. Acute and long-term toxicity tests were carried out by means of gross observation and weighing of inoculated animals. The distribution and retention time of plasmid F1-V/pVAX1 in mouse tissues were detected by PCR, and the antinuclear antibodies were detected by ELISA and ELISPOT methods. The safety of naked plasmid F1-V/pVAX1 was preliminarily verified, which provided the basis for the further study of oral live vector DNA vaccine. 4. The gene encoding F1-V fusion antigen was cloned into eukaryotic expression vector asd-pVAX1, into asd- Escherichia coli X6212, then transformed into asd- attenuated Salmonella X3730 X4550 in turn to obtain recombinant attenuated Salmonella X4550 (F1-V/AP). Recombinant plasmid was extracted and transfected into COS-7 cells. Immunohistochemistry and Western-blot were used.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 宋勤葉,楊漢春;核酸疫苗研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2004年03期
2 RobertsonJS,徐冰;核酸疫苗的安全性問題[J];國外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);1995年03期
3 何曉嬙 ,陳光明,黃英,楊富強(qiáng),吳樂園,莫國玉;治療型雙質(zhì)粒HBV DNA疫苗的構(gòu)建及其鑒定[J];解放軍醫(yī)學(xué)雜志;2003年06期
4 莫國玉 ,陳光明,黃芝瑛,何曉嬙,黃英,楊富強(qiáng);治療型HBV DNA疫苗在小鼠組織中的分布和長(zhǎng)期毒性研究[J];解放軍醫(yī)學(xué)雜志;2003年06期
5 莫國玉 ,陳光明,楊富強(qiáng),何曉嬙,黃英,楊若才;治療型HBV DNA疫苗肌注電轉(zhuǎn)染法免疫恒河猴的效果及安全性觀察[J];解放軍醫(yī)學(xué)雜志;2003年06期
6 陳東立,馬清鈞;表達(dá)霍亂CT-B和LPS-O抗原的鼠傷寒菌苗株的構(gòu)建[J];生物工程學(xué)報(bào);1997年01期
7 靳彥文;DNA疫苗接種的安全性[J];生物技術(shù)通訊;2001年01期
8 王忠澤,蔭俊,侯曉軍,宋偉,張松樂,王威,白潔;鼠疫耶爾森氏菌LcrV基因在大腸桿菌中的高效表達(dá)[J];生物技術(shù)通訊;2001年02期
9 傅林鋒,王秉翔,李啟明,雷清,蔣琳,沈心亮;鼠疫菌V抗原基因在大腸桿菌中的克隆及表達(dá)[J];微生物學(xué)報(bào);2003年04期
10 劉善奎,高申,鐘延強(qiáng),孫樹漢;DNA疫苗微球給藥系統(tǒng)的研究進(jìn)展[J];中國藥學(xué)雜志;2003年11期
，本文編號(hào)：2308621
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2308621.html
最近更新
教材專著
