Genistein对HIV-1gp120抑制鼠LTP的实验研究

目的为了探讨酪氨酸蛋白激酶抑制剂Genistein对人类免疫缺陷病毒Ⅰ型(HIV1)的包膜糖蛋白gp120引起大鼠海马脑片CA1区的长时程增强效应(Longtermpotentiation,LTP)作用的影响。方法应用离体脑片记录技术,记录大鼠海马CA1区的兴奋性突触后电位EPSP,研究了Genistein对gp120引起的大鼠海马脑片CA1区的突触传递和可塑性变化的影响。结果gp120对高频电刺激(HFS,100Hz,1000ms×2,串间隔20s,共2次)Schaffer侧支引起的大鼠海马CA1区LTP产生抑制作用,而对PTP没有影响。酪氨酸蛋白激酶抑制剂Genistein可以反转这种抑制效应。结论gp120可能通过抑制海马CA1区LTP而参与艾滋病痴呆(HIV1associateddementia,HAD)的形成,且这种抑制作用可能与酪氨酸蛋白激酶抑制剂Genistein有关。     

gp120mAb对gp120抑制大鼠海马脑片CA1区LTP作用的研究

目的探讨人类免疫缺陷病毒Ⅰ型(HIV-1)的包膜糖蛋白gp120特异抗体gp120mAb对gp120引起大鼠海马脑片CA1区的突触传递及可塑性变化的影响.方法应用离体脑片记录技术,记录大鼠海马CA1区的兴奋性突触后电位(EPSP),研究gp120mAb对gp120抑制高频电刺激Schaffer侧支引起的鼠海马长时程增强效应(LTP)作用的影响.结果gp120对高频电刺激(HFS,100 Hz,1 000 ms×2,串间隔20秒,共2次)Schaffer侧支引起的大鼠海马CA1区LTP产生抑制作用,而对其基础EPSP没有影响.用浓度为200 pmol/L的gp120灌流脑片,可引起LTP的维持发生抑制.这种抑制作用可被gp120特异抗体gp120mAb(50 ng/ml)所拮抗.结论gp120mAb可能是通过拮抗gp120抑制海马CA1区的LTP诱发和维持而参与艾滋病痴呆(HIV-1 associated dementia,HAD)的形成.     

原代HIV-1包膜糖蛋白修饰体诱导小鼠产生高滴度gp120抗体

目的 探索1型人类免疫缺陷病毒(HIV-1)包膜糖蛋白修饰对包膜免疫原性的的影响.方法 通过PCR扩增获得原代HIV-1 06044株包膜gp120基因及其突变体gp120/W427S基因,并构建gp120三聚体蛋白真核表达载体pcT-gp120和pcT-gp120/W427S,重组表达载体体外瞬时转染人胚肾HEK2...     

HIV-1包膜糖蛋白gp120对大鼠海马脑片电生理特性的影响

目的探讨人类免疫缺陷病毒I型(HIV-1)的包膜糖蛋白gp120对大鼠海马脑片CA1区神经元电生理特性及突触传递的影响。方法用盲法全细胞记录技术,观察gp120对大鼠海马脑片CA1区神经元电生理特性及对高频电刺激Schaffer侧支引起的鼠海马长时程增强效应(LTP)的影响。结果①在电流钳,gp120可使终末去极化电流激发快速动作电位的数目增加;②在电压钳,gp120对大鼠海马CA1区神经元的全细胞电流无明显作用;③将gp120(100 pmol/L)与海马脑片共孵育1h后,在钳制电压为-60 mV时,发现HFS后海马CA1区的兴奋性突触后电流(EPSC)显著减小,LTP的强度减少到(108.5±8.0)%(n=11,P<0.01)。结论gp120可使海马神经元的兴奋性增加,并可能通过抑制海马CA1区的LTP诱发参与艾滋病痴呆(HIV-1 associated dementia,HAD)的病理生理过程。     

Ⅰ型人类免疫缺陷病毒包膜gp120与乙型肝炎病毒表面抗原免疫原性比较

目的 比较Ⅰ型人类免疫缺陷病毒(HIV-1)及乙型肝炎病毒(HBV)包膜蛋白初次免疫及加强免疫后诱导产生抗体的规律,为提高HIV-1包膜蛋白诱导保护性抗体产生能力提供创新思路.方法 以10周龄雌性C57BL/6小鼠为动物模型,分别用HIV-1 06044毒株gp120三聚体(gp120T)、HBV表面抗原(HBsAg)蛋白与AddaVax佐剂免疫小鼠,背部皮下注射,共免疫3次,每次免疫间隔3周,第一、第二次免疫后7d和第三次免疫后3d、7d取血;第一次免疫后7d、第三次免疫后3d、7d取脾组织.用酶联免疫吸附实验(ELISA)及酶联免疫斑点实验(ELISpot)方法检测免疫小鼠血浆特异性结合抗体滴度及抗体分泌细胞(ASC)数量.结果 gp120T和HBsAg两种蛋白初次免疫后,动物均未产生明显的特异性抗体.两种蛋白加强免疫后特异性抗体水平明显升高,gp120T一次加强免疫及两次加强特异性抗体滴度逐渐升高,而HBsAg一次加强抗体滴度已经接近两次加强的水平.二次加强免疫后,gp120T和HBsAg免疫鼠脾脏特异性ASC数量差异不显著.结论 HIV-1包膜gp120T加强免疫诱导抗体水平达到高峰慢于HBsAg加强免疫,即加强免疫后gp120T诱导的回忆反应慢于HBsAg.     

含密码子优化型HIV-1 gp120基因重组腺病毒的构建及其免疫效果研究

目的 构建能表达野生型和密码子优化型人免疫缺陷病毒Ⅰ型(HIV-1)B亚型中国流行株gp120基因的非复制型腺病毒。方法 按哺乳动物细胞偏好的密码子对HIV-1B亚型中国流行株Ch gp42的gp120基因进行优化，合成优化基因。将野生型和密码子优化的gp120基因插入穿梭质粒，再与腺病毒骨架质粒pAdEasy-1共转化E．coli BJ5183，获得重组子，转染293细胞后获得重组病毒。分别以两种重组腺病毒疫苗免疫小鼠，ELISA检测小鼠血清中的特异性抗体，乳酸脱氢酶法检测小鼠细胞毒性T淋巴细胞(CTL)反应。结果 获得两株重组腺病毒rAd-wt．gp120和rAd．mod．gp120，能正确表达Gp120。rAd-mod．gp120比rAd-wt．gp120蛋白表达水平明显提高。重组腺病毒免疫小鼠后能产生HIV-1特异性的抗体及CTL反应，rAd-mod．gp120组明显优于rAd-wt．gp120组。结论 成功构建了表达野生型和密码子优化的HIV-1 gp120基因的重组腺病毒，能诱导HIV-1特异性体液和细胞免疫反应。     

共表达中国株HIV-1 gp120与hIL-6的重组鸡痘病毒研究

目的 构建共表达中国株HIV-1 gp120与人白细胞介素6(IL-6)的重组鸡痘病毒。方法 分别将HIV-gp120基因和hIL-6基因插入到鸡痘病毒表达载体pUTAL复合启动子ATI-P7．5和P7．5串联启动子下游，构建重组鸡痘病毒表达质粒pUTA-GP-IL6。利用脂质体法将重组质粒和鸡痘病毒282E4株共转染鸡胚成纤维细胞。经BUdR加压筛选3次后，重组病毒分别用PCR、间接免疫荧光试验和Western blot进行鉴定，并进行小鼠免疫研究。结果重组病毒基因组中可扩增出1．4kb大小片段，重组病毒感染细胞表面有绿色荧光物质，表达产物的Western blot分析表明重组病毒可表达gp120和hIL-6蛋白。重组病毒可刺激小鼠产生特异性体液免疫应答。结论成功构建了共表达中国株HIV-1 gp120与hIL-6的重组鸡痘病毒，为研制HIV-1基因工程活载体疫苗提供有益的资料。     

人免疫缺陷病毒Ⅰ型包膜糖蛋白gp120基因在大肠杆菌中的高效表达

目的提高人免疫缺陷病毒Ⅰ型（ＨＩＶ－１）包膜糖蛋白ｇｐ１２０基因在原核系统中的表达量。方法采用聚合酶链反应（ＰＣＲ）技术扩增出５６０ｂｐ的ＨＩＶ－１ＬＡＶ株ｇｐ１２０Ｎ端基因片段，经ＥｃｏＲⅠ及ＳａｌⅠ酶切后插入高效表达载体ｐＥＴ２８ａ，得到重组质粒ｐＥＴ１２０，并转化表达宿主菌ＢＬ２１（ＤＥ３），经诱导高效表达出ＨＩＶ－１ｇｐ１２０基因片段。结果间接酶联免疫吸附试验（ＥＬＩＳＡ）及Ｗｅｓｔｅｒｎｂｌｏｔ实验表明，表达产物具有良好的抗原性及特异性。ＳＤＳ－ＰＡＧＥ电泳分析结果表明，ｇｐ１２０表达量占总菌体蛋白的５０％。结论在原核系统中高效表达了ＨＩＶ－１ｇｐ１２０基因     

抗人免疫缺陷病毒1型gp120人源单链抗体的表达

目的研究人源抗人免疫缺陷病毒１型（ＨＩＶ－１）ｇｐ１２０单链抗体（ＳｃＦｖ）。方法以人工合成的ＨＩＶ－ｌｇｐ１２０Ｖ３环多肽为抗原，利用噬菌体抗体库技术，筛选含有抗－ｇｐ１２０ＳｃＦｖ基因的噬菌体，提取质粒，转化大肠杆菌ＨＢ２１５１，表达可溶性ＳｃＦｖ。结果经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ分析，表达产物分子量为２８ｋＤ左右，且具有ｃ－ｍｙｃ活性；ＥＬＩＳＡ和Ｄｏｔｂｌｏｔ结果表明，可溶性ＳｃＦｖ具有较好的抗原结合活性和较强的特异性；竞争性ＥＬＩＳＡ实验结果进一步证明表达产物的特异抗－ｇｐ１２０活性。结论该技术便捷有效，可大量获得人源抗ＨＩＶ抗体片段，为进一步研究抗ＨＩＶ抗体的生物活性和ＨＩＶ感染诊治打下基础     

HIV-1包膜蛋白gp120在神经元损伤诱发认知障碍中的作用

目的探索HIV-1包膜蛋白gp120在神经元损伤引起认知障碍中的作用。方法免疫印迹和免疫荧光检测gp120处理后的小胶质细胞活化、炎症因子表达和神经元损伤情况;免疫组化分析gp120转基因小鼠的神经元损伤情况;行为学分析转基因小鼠的神经认知状况。结果体内和体外试验结果表明HIV-1 gp120可显著诱导caspase-1与IL-1β表达,并间接引起神经元突触变短和神经元损伤(P<0.05);与野生型小鼠相比,gp120转基因小鼠出现明显的皮层和海马回小胶质细胞激活、神经元丢失与树突损伤以及神经认知紊乱现象。结论HIV-1 gp120可能通过激活小胶质细胞炎症因子释放导致神经元损伤并可能与神经认知障碍发生相关。     

HIV-1 gp120 AND IMMUNE NETWORK

It has been demonstrated that the immunodominant V3 loop of HIV-1 gp120 and its flanking regions bear sequence and structural homology to the framework and complementarity-determining regions of human immunoglobulins. It has been proposed that the Ig-like domain of gp120 might encode idiotypes and in this way permit HIV-1 entry into the immune regulatory network. This notion is strongly supported by results demonstrating that the anti-V3 loop and anti-Ig antibodies of healthy individuals share complementary structure and that V3 reactive antibodies are present in HIV-negative sera. This might be the mechanism by which HIV induces immunological abnormalities, and it should be taken into consideration in AIDS vaccine development.     

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN-gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN-gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN-gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.     

RECOMBINATION PROPERTY OF THE HIV-1 gp120 GENE

Using a chimeric primer consisting of the nucleotide sequence derived from the HIV-1 envelope gene coding for the second conserved region of gp120, and the highly conserved sequence derived from the human immunoglobulin gene coding for the V_HIII domain, it has been identified in sera of AIDS patients HIV-1 field isolates carrying the complete and active Chi recombination hot spot (GCTGGTGG). The recombination between the HIV-1 gene coding for the central portion of gp120 and the bacterial gene coding for the clp protease was also demonstrated in vivo. These results point out serious concern that vectored AIDS vaccine candidates carrying the HIV-1 env gene on viral and bacterial vectors could become the source of potentially new infectious diseases rather than an effective instrument for AIDS prevention.     

HIV-1糖蛋白gp120激发人小胶质细胞钙内流效应和ERK磷酸化

目的： 探讨HIV-1糖蛋白gp120对人小胶质细胞钙离子内流和ERK磷酸化的作用及其机制。方法: 用钙离子探针Fluo-4标记粘附在盖玻片上的人小胶质细胞，运用共聚焦显微镜以荧光强度为指标实时观察各种条件下的细胞内钙离子水平的变化；用gp120处理并用anti-gp120-FITC进行染色，运用共聚焦显微镜术和流式细胞术分析人小胶质细胞与gp120结合情况；用抗磷酸化ERK 抗体免疫荧光方法进行染色，运用共聚焦显微镜术和流式细胞术进行ERK磷酸化水平分析。结果: 共聚焦显微镜检测结果显示HIV-1 糖蛋白gp120能够激发人小胶质细胞钙离子内流效应；共聚焦显微镜和流式细胞仪分析结果显示gp120可以与人小胶质细胞结合；共聚焦显微镜和流式细胞仪分析结果显示gp120刺激可增加人小胶质细胞ERK磷酸化。结论: HIV-1 糖蛋白gp120能在人小胶质细胞激发钙离子内流并且增加胞内ERK的磷酸化，从而导致了小胶质细胞的活化，这一效应提示，在HIV-1相关性脑炎中，gp120可能参与了某些发病机制。     

Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120

In order to improve the immunogenicity of HIV-1 envelope glycoproteins, we have fused gp120 to a carrier protein, hepatitis B surface antigen (HBsAg), which is capable of spontaneous assembly into virus-like particles. The HBsAg-gp120 hybrid proteins assembled efficiently into 20-30 nm particles. The particles resemble native HBsAg particles in size and density, consistent with a lipid composition of about 25% and a gp120 content of about 100 per particle. Particulate gp120 folds in its native conformation and is biologically active, as shown by high affinity binding of CD4. The particles express conformational determinants targeted by a panel of broadly cross-reactive neutralizing antibodies, and they show tight packing of gp120. Because the particles are lipoprotein micelles, an array of gp120 on their surface closely mimics gp120 on the surface of HIV-1 virions. These gp120-rich particles can enhance the quality, as well as quantity, of antibodies elicited by a gp120 vaccine.     

An anti-HIV-1 gp120 antibody expressed as an endocytotic transmembrane protein mediates internalization of HIV-1

In this study, we used HIV-1 as a model to demonstrate a novel approach for receptor-independent cell entry of virus. The heavy chain of an anti-HIV-1 gp120 antibody was engineered with endocytotic and transmembrane motifs from either the cation-independent mannose 6-phosphate receptor or the low-density lipoprotein receptor. Flow cytometry and immunofluorescence studies showed that the chimeric antibodies were expressed on the cell surface and can undergo rapid internalization. Furthermore, one of the chimeric antibodies was able to bind and internalize HIV-1. Using a luciferase reporter HIV-1, we further showed that internalized viruses could undergo replication. Therefore, we have demonstrated a proof-of-principle of a novel method that can be used to internalize virus into cells, without prior knowledge of the cellular receptor for the virus. We propose that this approach would be particularly useful for studying viruses whose cellular receptor(s) is not known.     

A versatile vector for the production of pseudotyped viruses expressing gp120 antigens from different clades of primary HIV-1 isolates

A novel HIV-1 Env expression vector (SF162-Z) was developed by introducing two new cloning sites on the backbone of an existing vector that produces a full length Env from HIV-1 SF162 isolate. These sites facilitate the swapping of the gp120 portion of the SF162 Env with matching gp120 antigens from HIV-1 isolates of different genetic clades. Final production of functional pseudotyped viruses will express chimeric Env antigens, including gp41 of the parental SF162 and gp120 from other primary isolates. This system is useful for testing the neutralizing sensitivity of partial env gene products frequently identified in viral quasi species in patients infected with HIV or when only partial gp120 gene products are available.