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hiv-2gp105-gag截短体在毕赤酵母中重组表达研究

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中华微生物学和免疫学杂志 2003年第5期23卷 371-374页

作者：李子健金宁一江文正张应玖张立树王宏伟吉林大学第一医院解放军军需大学研究所解放军基因工程重点实验室吉林大学生命科学学院

目的　实现hiv 2 RODgp10 5 gag截短体重组蛋白tgp10 5 gag的高效分泌表达 ,对其表达条件进行研究。方法　用PCR方法获得hiv 2 RODtgp10 5截短基因 ,将其与hiv 2 RODgag构建hiv 2 RODtgp10 5 gag嵌合基因并克隆到毕赤酵母 (Pichiapas... 详细信息

目的　实现hiv 2 RODgp10 5 gag截短体重组蛋白tgp10 5 gag的高效分泌表达 ,对其表达条件进行研究。方法　用PCR方法获得hiv 2 RODtgp10 5截短基因 ,将其与hiv 2 RODgag构建hiv 2 RODtgp10 5 gag嵌合基因并克隆到毕赤酵母 (Pichiapastoris)分泌型表达载体pPIC9中 ,转化GS115酵母菌 ,经MD/MM表型筛选、PCR扩增筛选阳性克隆 ,以甲醇诱导表达后进行SDS PAGE分析及Westernblot证实。结果　hiv 2 RODtgp10 5 gag嵌合截短基因在Pichiapastoris酵母系统中获得了有效分泌表达 ,相对分子质量 (Mr)约为 14 0× 10 3 ,表达量约为 16 % ,表达产物可被hiv 2阳性血清识别。最佳表达条件是大于 85 %的溶解氧 ,摇瓶转速 2 4 0r/min ,1.0 %～ 1.5 %甲醇诱导 ,培养时间 3d。结论　在Pichiapas toris表达系统中实现了hiv 2 RODtgp10 5 gag蛋白的有效分泌性表达 ,表达产物具有良好的抗原特异性。

关键词： hiv-2 人免疫缺陷病毒 gpl05-gag 毕赤酵母基因表达

小鼠对hiv-2gp105核酸疫苗免疫应答的研究

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细胞与分子免疫学杂志 2005年第1期21卷 86-89页

作者：李子健金宁一江文正张立树军事医学科学院军事兽医研究所全军基因工程重点实验室

　目的: 探讨hiv- 2gp105基因核酸疫苗在小鼠体内的免疫应答, 为开发hiv- 2核酸疫苗提供实验依据。方法:将hiv- 2外膜蛋白 (gp105 )基因插入真核表达质粒载体pVAX1中, 构建pVAX1 gp105重组表达质粒。将其肌注免疫BALB/c小鼠, 用ELISA法... 详细信息

　目的: 探讨hiv- 2gp105基因核酸疫苗在小鼠体内的免疫应答, 为开发hiv- 2核酸疫苗提供实验依据。方法:将hiv- 2外膜蛋白 (gp105 )基因插入真核表达质粒载体pVAX1中, 构建pVAX1 gp105重组表达质粒。将其肌注免疫BALB/c小鼠, 用ELISA法检测小鼠血清抗hiv -2抗体, 用流式细胞仪测定CD4+、CD8+ T细胞亚群数, 以乳酸脱氢霉释放法检测脾特异性CTL的杀伤活性。结果: 重组质粒pVAX1 -gp105免疫组小鼠的血清抗体滴度、脾T细胞亚群的数量及特异性CTL的杀伤活性, 均明显高于对照组, 分别为Phiv -2gp105核酸疫苗能诱导小鼠产生特异性细胞和体液免疫。

关键词： hiv-2 gp105 DNA疫苗免疫应答

hiv-2 gp105重组鸡痘病毒引起小鼠细胞和体液免疫应答

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中华微生物学和免疫学杂志 2005年第10期25卷 821-822页

作者：张立树金宁一李子健江文正宋英今王宏军事医学科学院军事兽医研究所全军基因工程重点实验室长春130062 北京大学第三医院血管医学研究所第二军医大学免疫学研究所暨南大学生命科学院生物系

人免疫缺陷病毒Ⅱ型(human immunodeficiencyvirus type 2,hiv-2)于1986年在几内亚-比绍被发现.起初hiv-2仅在西非呈区域性流行,随后在欧洲、美洲和亚洲也出现了感染者和发病者.我国的首例hiv-2感染者于1998年在福建省发现[1],并检测出H... 详细信息

人免疫缺陷病毒Ⅱ型(human immunodeficiencyvirus type 2,hiv-2)于1986年在几内亚-比绍被发现.起初hiv-2仅在西非呈区域性流行,随后在欧洲、美洲和亚洲也出现了感染者和发病者.我国的首例hiv-2感染者于1998年在福建省发现[1],并检测出hiv-1与hiv-2的共感染患者[2].因此,研究有效的hiv-2疫苗势在必行.hiv-2 env基因编码前体蛋白gp140,后者裂解后生成外膜蛋白gp105和跨膜蛋白***105是病毒的主要抗原,其表面具有大量的Ab表位、TH表位和CTL表位,能诱导机体产生强烈而广泛的中和抗体和细胞毒(CTL)反应,因此是基因工程疫苗研究的重要靶点.本研究将已构建的hiv-2 gp105重组鸡痘病毒大量制备后免疫BALB/c小鼠,观察其在小鼠体内的免疫原性,以获得第一手的实验资料。

关键词： BALB/c小鼠重组鸡痘病毒 hiv-2 gp105 体液免疫应答 immunodeficiency 细胞毒人免疫缺陷病毒Ⅱ型 CTL表位疫苗研究

hiv-2gag基因在重组痘苗病毒中的表达

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中国兽医学报 1997年第6期17卷 570-574页

作者：王秀清金宁一田梅毛春生李体远王宏伟苏忠兰殷震解放军农牧大学军事兽医研究所白求恩医科大学应用基础医学研究所白求恩医科大学预防医学院

以痘苗病毒ＨＡ基因为侧翼，将ＨＩＶ－２ｇａｇ基因部分序列（简称Ｇ１）和全部序列（简称Ｇ２）分别插入牛痘病毒Ａ型包涵体（ＡＴＩ）和串联１０个（与ＨＡ基因反向）或１６个（与ＨＡ基因同向）痘苗病毒Ｐ７．５组成的复合型启动子... 详细信息

以痘苗病毒ＨＡ基因为侧翼，将ＨＩＶ－２ｇａｇ基因部分序列（简称Ｇ１）和全部序列（简称Ｇ２）分别插入牛痘病毒Ａ型包涵体（ＡＴＩ）和串联１０个（与ＨＡ基因反向）或１６个（与ＨＡ基因同向）痘苗病毒Ｐ７．５组成的复合型启动子下游，构建了４个重组痘苗病毒表达载体质粒。经脂质体转染、鸡红细胞吸附试验筛选和免疫荧光鉴定，获得４株能稳定表达目的蛋白的重组痘苗病毒。实验结果表明，以ＨＡ基因为侧翼构建重组病毒的重组率约为０．１％，阳性重组率为２５％～１００％。实验中还发现，以ＨＡ基因为侧翼的重组痘苗病毒能够引起细胞融合，其可作为筛选重组痘苗病毒的另一种标志。Ｗｅｓｔｅｒｎｂｌｏｔ结果表明，表达的Ｇａｇ蛋白能被ＨＩＶ－２血清中的特异性抗体所识别，并能诱导小鼠产生抗ＨＩＶ－２Ｇａｇ抗体。

关键词： hiv-2 gag基因痘苗病毒表达

Laboratory Profile of hiv-2 and Dual hiv-1/hiv-2 Associated Acquired Immunodeficiency Syndrome in Nigeria

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World Journal of AIDS 2013年第3期3卷 192-196页

作者： Georgina Njideka Odaibo David Olufemi Olaleye Department of Virology College of Medicine University of Ibadan University College Hospital Ibadan Nigeria.

Background: hiv-2 is comparatively less pathogenic with slow progression of infection to clinical disease and consequently there is less of information on the occurrence of hiv-2 associated disease than hiv-1. We hereby describe some laboratory profiles of individuals presenting with hiv-2 and dual hiv-1/2 related AIDS at the University College hospital in Ibadan over a period of seven years. Methodology: Blood samples from patients presenting with the AIDS defining illness at the University College Hospital, Ibadan, Nigeria were tested for antibodies to hiv-1/2 using rapid test devices or ELISA. Initially reactive samples were further tested by immunoblotting for differentiation into hiv-1 or hiv-2 or hiv-1/2 dual infection. Blood samples from individuals with confirmed infections were further analyzed for CD4 cell lymphocyte number, plasma hiv-1 RNA concentration, hematological and blood chemistry parameters. The data analysis was done using descriptive statistics and Levene-S test for equality of variance. Results: Thirty five patients, 18 and 17 with hiv-2 and dual hiv-1/2 infections respectively were identified during the period covered by this study (2005-2012). The median age of the patients was 48 years old (Range: 42 - 70 years old) and mean CD4 cell count of hiv-2 patients at enrollment was 324 (Range: 16 - 696) and 350 (Range 54 - 863) per microlitre of blood for patients with dual hiv-1/2 infection. hiv-1 RNA was not detected in the plasma of the 18 patients with serological hiv-2 infection but 2 (11.8%) of the 17 patients with dual hiv-1/2 serological profile had detectable hiv-1 RNA (1,287,275 copies/ml and 1,816,491 copies/ml). Conclusion: The results emphasize the need to consider hiv-2 infection in the investigation of patients presenting with the AIDS related illness but with negative hiv-1serology. The study also shows the importance of inclusion of multispot hiv-1 and 2 rapid tests for differentiating hiv-1 from hiv-2 infections in regions where

关键词： hiv-2 hiv1/2 Dual Infection CD4 Hematology Chemistry Nigeria

Trends in hiv-2 Seroprevalence at the National Reference Center of hiv from 2005 to 2014 in Lome, Togo

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World Journal of AIDS 2017年第4期7卷 239-246页

作者： Amivi Amenyah- Ehlan Mounerou Salou Malewe Kolou Komlan Ali- Edje Tufa Nyasenu Sika Dossim Alassane Ouro- Medeli Mensah Douffan Anoumou Dagnra Mireille Prince- David Laboratoire de Biologie Moleculaire et d’immunologie de la Faculte des Sciences de la Sante de l’Universite de Lome (BIOLIM/FSS-UL) Lome Togo Centre National de Reference Pour les Tests VIH et les IST (CNR-VIH) Lome Togo

Background: In Togo, the hiv/AIDS epidemic is characterized by the circulation of the 2 subtypes of hiv. Thus, patients infected with hiv-2 are diagnosed and monitored in the care centers. Objective: To document the trend of hiv-2 prevalence over a decade of activities of the National Reference Center for hiv tests and screening (CNR-VIH). Methods: A cross sectional study was carried out from the screening data archived from January 2005 to December 2014 at the CNR-VIH, a laboratory located in the Sylvanus Olympio University Hospital (CHU SO) Lome. The sampling consisted of adults and children outpatients or those who were hospitalized in CHU SO, subjects presenting for voluntary testing, pregnant women and patients or samples referred for hiv confirmation. All samples were tested for hiv-1 and hiv-2 infections by combining ELISA and rapid diagnostic tests. Result: During the decade, 34,077 subjects were screened for hiv infection. The overall prevalence of hiv infection was 20.70% (7055/34077). In 10 years, the prevalence of hiv infection in CNR-VIH decreased significantly from 35.40% CI95% [34.50% - 36.20%] in 2005 to 14.20% CI95% [13.60% - 14.70%] in 2014 (p = 0.03). The prevalence of hiv-1, hiv-2 and dual hiv1&hiv-2 was respectively 20.40%, 0.23% and 0.07%, with annual prevalence of hiv-2 between 0.07% and 0.39%. The differences between the hiv-2 prevalence over the decade were not statistically significant (p > 0.15). A 4-year-old child from mother-to-child hiv-2 transmission was diagnosed. Sixty-five percent of adult patients were over 40 years of age with an average age of 43.5 ± 11.3 years. Conclusion: Data from the National Reference Center for hiv Tests in Togo over the last ten years confirm the existence of a weak epidemic of hiv- 2 infection with a tendency towards stability.

关键词： hiv-2 Seroprevalence Togo

hiv-2 env基因在大肠杆菌和重组痘苗病毒中的表达

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中国兽医学报 1998年第3期18卷 216-220页

作者：王秀清金宁一田梅郭志儒方厚华顾万钧李萍殷震解放军农牧大学军事兽医研究所白求恩医科大学应用基础医学研究所白求恩医科大学预防医学院

将编码人Ⅱ型免疫缺陷病毒（ＨＩＶ－２）ｅｎｖｇｐ１２０（Ｅ１）和ｇｐ３６（Ｅ２）分别克隆到原核高效表达载体ｐＥＴ１７ｂ、ｐＢＶ２２０和真核表达载体ｐ１０、ｐ１６中，构建成８个重组质粒。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒ... 详细信息

将编码人Ⅱ型免疫缺陷病毒（ＨＩＶ－２）ｅｎｖｇｐ１２０（Ｅ１）和ｇｐ３６（Ｅ２）分别克隆到原核高效表达载体ｐＥＴ１７ｂ、ｐＢＶ２２０和真核表达载体ｐ１０、ｐ１６中，构建成８个重组质粒。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ分析证明，在原核表达系统成功地表达了ＨＩＶ－２ｅｎｖ融合蛋白，表达的蛋白分子量分别为３５０００、７００００和５００００，而原核表达质粒ｐＥＴＥ１在ＳＤＳ－ＰＡＧＥ凝胶的３５０００处可见明显的额外蛋白带。经薄层扫描测定，原核系统表达的Ｅｎｖ蛋白（ｇｐ１２０）相对含量为５．８％。在真核细胞中表达的Ｅｎｖ蛋白经Ｗｅｓｔｅｒｎｂｌｏｔ检测分析，分子量分别为６００００、５７０００和４２０００。上述表达的Ｅｎｖ蛋白均能与ＨＩＶ－２阳性血清发生特异性反应。

关键词： hiv-2 Env蛋白表达大肠杆菌痘苗病毒

Higher Type 1 Interferon Levels in Plasma of Asymptomatic hiv-2 than in hiv-1 Individuals

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Advances in Infectious Diseases 2016年第1期6卷 15-23页

作者： Samuel V. Nuvor Hilton Whittle Sarah Rowland-Jones Assan Jaye MRC Laboratories Fajara Banjul The Gambia School of Medical Sciences University of Cape Coast Cape Coast Ghana London School of Hygiene & Tropical Medicine London UK MRC Human Immunology Unit Weatherall Institute of Molecular Medicine John Radcliffe Hospital Oxford UK Hospital A. le Dantec Laboratoire de Bacteriologie Virologie (LBV) Universite Cheikh Anta Diop (UCAD) Dakar Senegal

A number of cytokines are secreted during hiv infection that enhances both innate and adaptive immune responses. Interferon-α/β/γ, IL-12, IL-15 and IL-18 have been found to contribute to the development, maturation, differentiation and function of NK and other immune cells. The levels of IFN-α/β/γ, IL-12, IL-15 and IL-18 were compared in the plasma of 90 hiv-1 infected and 90 hiv-2 infected subjects by ELISA or Cytometric Beads Array assays. The hiv-infected subjects were stratified according to CD4+ T cell counts into three groups: >500, 200 - 500 and hiv uninfected blood bank donors. Among the cytokines tested, IFN-α was found to be significantly increased in hiv-2 infected compared to hiv-1 infected subjects at high CD4+ T cell counts (p = 0.001). The levels of IFN-β were seen to differ between the two infections in patients from the category of medium CD4+ T cell counts: this was significantly increased in hiv-2 infected patients (p + T cell categories except for an increase in hiv-2 infected patients at low CD4+ T cell counts (p = 0.02). The levels of these cytokines were similar in all hiv-1 subjects. Also, the level of IL-12p70 was similar between the two infections but significantly higher in hiv-2 at low compared to medium CD4+ T cells categories (p = 0.047). Similar to IFN-γ and IL-12p70, the levels of both IL-18 and IL-15 were found to be significantly higher in hiv-2 infected patients compared to hiv-1 at low CD4+ T cell counts (p hiv infection but the finding of increased IFN-α in hiv-2 infected asymptomatic subjects is consistent with the high innate NK respo

关键词： Type I Interferon hiv-I hiv-2 Cytokines Interleukins

hiv-2DNA疫苗免疫小鼠的免疫反应观察

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免疫学杂志 2002年第5期18卷 333-336页

作者：张应玖金宁一郭焱江文正李子健沈家骢吉林大学生命科学学院吉林长春130023 解放军军需大学解放军基因工程重点实验室吉林长春130062

目的　用构建的hiv 2外膜蛋白gp1 0 5和核心蛋白gag基因的DNA疫苗免疫小鼠 ,评价其免疫效果。方法　将hiv 2型gp1 0 5和gag基因克隆到表达载体pIRES1neo中 ,构建重组DNA疫苗质粒。间接免疫荧光试验证明 ,构建的DNA疫苗在真核细胞中表达... 详细信息

目的　用构建的hiv 2外膜蛋白gp1 0 5和核心蛋白gag基因的DNA疫苗免疫小鼠 ,评价其免疫效果。方法　将hiv 2型gp1 0 5和gag基因克隆到表达载体pIRES1neo中 ,构建重组DNA疫苗质粒。间接免疫荧光试验证明 ,构建的DNA疫苗在真核细胞中表达了gp1 0 5或 /和gag。构建的疫苗免疫小鼠后 ,用流式细胞仪测定CD4+ 、CD8+ T淋巴细胞亚类数 ,并用hiv 2抗体ELISA检测试剂盒检测免疫鼠血清中抗hiv 2抗体水平。结果　构建了 3种hiv 2DNA疫苗pIRES1gag、pIRES1gp1 0 5和pIRES1gag gp1 0 5 ,转染BHK细胞后均能表达抗原蛋白 ,免疫小鼠后可有效地刺激淋巴细胞增殖并诱导产生抗hiv 2特异性抗体 ,其中pIRES1gag gp1 0 5免疫鼠中 ,淋巴细胞增殖最显著 ,而pIRES1gp1 0 5免疫鼠中hiv 2特异性抗体水平最高。结论　构建的DNA疫苗均能诱导机体产生免疫反应 ,其中pIRES1gp1 0 5诱导的体液免疫应答最显著 ,而pIRES1gag gp1 0 5诱导的细胞免疫响应最显著。

关键词：免疫应答艾滋病 hiv-2 DNA疫苗小鼠