To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type(HIV-1)transmembrane protein gp41 epitopes and the disease progression,it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes.For this purpose we evaluated monoclonal antibodies(mAbs)against the gp41 core structure such as mAbs 50.69,98.6 and T26,byWestern blotting(WB)and flow cytometry.WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41,and mAb T26 exclusively bound to oligomeric gp41.We evaluated the sera from Pneumocystis pneumonia patients(PCP;n=7)and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry.The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs,and the PCP sera inhibited mAb T26 binding more efficiently than LTS.Therefore,PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure(binding sites of mAbs 50.69 and 98.6)but also against oligomeric gp41 specific epitope(binding site of mAb T26).

To elucidate the roles of serine proteases,including thrombin,in HIV infection,we treated H9 cells infected with HIV-1 LAI virus(H9/ⅢB)with four different proteases(thrombin,cathepsin G,trypsin and chymotrypsin)and observed their effects on functional epitopes on both gp120 and gp41 by using flow cytometry.Monoclonal antibodies(MAbs)against the V3 loop,V2 loop,CD4 binding site,coreceptor binding site and gp41 were used.It was found that trypsin decreased the binding of all MAbs except for one MAb against the V3 loop(ⅢB-V3-21).Chymotrypsin and cathepsin G did not show any remarkable effect on the antigen expression.On the other hand,thrombin decreased the reactivities of two out of four anti-V3 MAbs and increased the exposure of functional gp120 epitopes including the coreceptor binding site and CD4 binding site.Thrombin also increased the expression of 2F5 antigen(a neutralizing epitope of gp41)but had no effect on other gp41 epitopes.The effect of trypsin or thrombin on HIV-induced cell fusion was examined through co-culturing H9/ⅢB and MAGI cells.Trypsin slightly inhibited fusion.Fusion was significantly enhanced in a dose-dependent manner by thrombin,and a 280% increase at 5U/ml(P<0.001)was observed.In conclusion,thrombin,one of the major inflammatory molecules in blood,facilitates HIV-induced cell fusion,probably by activating gp120.

V3 loop peptides from three different human immunodeficiency virus type 1(HIV-1)strains were synthesized.BH10,ADA,and 89.6 strains whose infections are dependent on CXCR4,CCR5,and both,respectively, were selected.Co-transfection of luciferase reporter gene and corresponding envelope genes (HXB2, ADA, and 89.6) generate pseudotype viruses (HXB2/Luc, ADA/Luc, and 89.6/Luc). The effects of each peptide on the infection of U87 cells expressing CD4 and one of the coreceptors with all pseudotype viruses were evaluated. V3 loop peptide from BH10(V3-BH10)alone increased the HXB2/Luc infection by 93% at 10lM. Both V3-ADA and V3-89.6 enhanced ADA/Luc infection by 38% and by 55% at 10lM,respectively.For 89.6/Luc infection,only V3-89.6 enhanced the infections on both target cells.V3-BH10 modulated the epitopes of coreceptor binding site and V2 loop of gp120 on HIV-1 ⅢB infected H9 cells, indicating that V3 loop peptide activates viral gp120 and enhances infectivity.

Nine different DNA enzymes(DzV3-n,n=1-9)targeting the V3 loop region of HIV-1 HXB2 were synthesized. One of those, DzV3-9, efficiently cleaved the target in the conserved sequence in the RNA transcript in vitro.DzV3-9 was stable in the cells and inhibited replication of both NL432 and SF162 strains in U87 cells expressing CD4 and co-receptors. The inhibitory effect of DNAzyme on incoming HIV-1 was also demonstrated with pseudotype virions generated by NL432-based luciferase reporter genes.Thus,an efficient,stable DNAzyme against a functionally important region of HIV-1 was identified,and it may be useful for prevention of HIV-1 infection.

目的　分析黑龙江省Ⅰ型人免疫缺陷病毒（human immunodeficiency virus type 1，HIV-1）原代分离株包膜糖蛋白的变异性及表型特征。方法　从一名HIV 阳性但未发病的感染者外周血单个核细胞提取DNA，采用保守引物进行HIV 包膜直接克隆，进行序列分析及系统树分析。构建了一株带该包膜蛋白的伪病毒并利用表达CXCR4 和CCR5 的靶细胞进行了感染实验，观察该病毒包膜利用辅助受体的表型特征。结果 共获得2个有功能全长env克隆，分别命名为CHNHLJ03009c34（Gen2Bank 序列号为AY905493）和CHNHLJ03009c33。用全长env氨基酸序列与国内外分离株进行同源性比较分析发现，与分离株CHNHLJ03009c34 的包膜同源性最高的病毒为云南的HIV21 B'亚型分离株RL42，同源性为91.52%。系统发育分析结果表明，该株为泰国B'亚型，与云南分离株RL42的遗传距离最近。对包膜蛋白结构分析结果显示，分离株CHNHLJ03009c34包膜在抗原性和亲水性上与RL42没有明显差别。感染性检测结果显示该病毒只能感染U87. CD4. CCR5细胞，不能感染U87.CD4. CX2CR4 细胞。结论 本研究从黑龙江省一名HIV-1阳性者克隆到HIV-1 CHNHLJ03009病毒的包膜基因，该病毒属于B'亚型（泰国亚型），为R5亲嗜性毒株。该包膜基因克隆为首次报道的黑龙江分离株。

目的　构建V3 环缺失的HIV-1 ADA 株包膜糖蛋白表达体系。方法　分别设计包膜糖蛋白两端及V3 环两端的两对引物，采用重叠延伸剪接法，进行HIV-1 ADA 株包膜糖蛋白V3 环缺失体的构建。得到的PCR 产物经EcoR Ⅰ和Xho Ⅰ双酶切，与pSM载体连接，构建HIV-1 ADA 株包膜糖蛋白V3 环缺失的表达载体（pSM-ADAΔV3），转化大肠杆菌后筛选阳性克隆，经PCR及基因序列测定进行鉴定。结果　获得HIV-1 ADA 株包膜糖蛋白V3 环缺失体PCR 产物，构建了其表达载体pSM2ADAΔV3 ，经转化和筛选获得了重组菌，经PCR 及基因测序结果显示重组质粒序列正确，为预期目的片段。结论　成功构建了V3 环缺失的HIV-1 ADA 株包膜糖蛋白的表达载体。此结果为进一步构建伪病毒，观察V3 环完全或部分缺失对病毒侵入靶细胞过程的影响，为开发阻止HIV-1 进入靶细胞的药物或疫苗打下基础。

目的 明确Ⅰ型人免疫缺陷病毒（human immunodeficiency virus type 1 ，HIV1）包膜糖蛋白gp120第三可变区（variable region ，V3） 在病毒进入靶细胞的早期阶段中的作用。方法　合成了3 个环状V3多肽，包括V3 BH10、V3 ADA 和V3 89. 6 以及直链和短链多肽V3 BH10/ CA、V3 NNT24 和V3 IRI12。构建了3株分别带HIV HXB2 株、ADA 株和89. 6 株包膜的伪病毒并观察了多肽对不同嗜性HIV 侵入靶细胞能力的影响。结果 （1）HIV1V3区多肽以毒株特异性模式增强HXB2、ADA 和89.6进入靶细胞;（2） 直链V3多肽与其环状多肽具有相似作用;带有C末端的短肽V3NNT24也有与长链V3 BH10/ CA 相近的作用，只有中央保守区的短链V3 IRI12 没有类似功能。结论　本研究首次发现HIV1V3区在病毒进入靶细胞早期阶段中具有毒株特异性增强病毒侵入的作用。该发现有助于进一步明确HIV1进入靶细胞的机制。

目的：研究人类免疫缺陷病毒（HIV）不同亲嗜株包膜糖蛋白V3区结合于靶细胞的能力。方法：合成来源于不同嗜性HIV-1V3区的生物素标记和非标记的多肽。采用流式细胞计数分析生物素化的V3多肽对细胞的结合能力以及细胞表面的结合配体。结果：HIV-1 X4亲嗜株ⅢBV3区能结合于多种细胞的表面，包括辅助受体CXCR4；竞争实验结果显示蛋白酶抑制剂能抑制该结合。Rs亲嗜株ADA V3区只极微弱地结合于外周血单核细胞和表达CCRs的人星形胶质细胞表面。结论：不同嗜性HIV-IV3区结合于细胞表面的能力不同，X4 亲嗜株V3区直接结合于细胞表面并被其自身所增强，其靶分子至少包括辅助受体CXCR4和蛋白酶分子；而心亲嗜株ADA V3区则不结合于CCR5和蛋白酶。

目的 研究腺病毒介导的野生型P53基因（Ad-P53）抗人胃腺癌细胞增殖的效果。方法 利用携带P53基因的复制缺陷型腺病毒（Ad-P53）感染人胃腺癌细胞株SGC-7901，以携带报告基因LacZ的同型载体（Ad-LacZ）作对照，MTT法测定及克隆形成实验分析P53基因对胃腺癌细胞的体外抑瘤效应。用流式细胞技术分析P53基因转染后对细胞周期的影响以及引起凋亡情况，并用电镜观察细胞的超微结构变化。结果 野生型P53基因转移对SGC-7901具有明显的抗增殖作用。Ad-P53感染SGC-7901后细胞生长明显被抑制，抑制率达88.73%（MOI为200，7d）。克隆形成能力也明显受到抑制，当MOI分别为50，100，200时，克隆形成数为22，0，0。流式细胞计数分析显示.Ad-P53（能引起SGC-7901细胞发生周期阻滞和细胞凋亡，凋亡百分率达71.4%（MOI为200，7d）。电镜观察结果也显示细胞出现凋亡的特征性形态改变。结论 腺病毒成功介导野生型P53基因转移入人胃腺癌SGC-7901细胞中，野生型P53基因有显著抑制胃腺癌细胞增殖的作用。其表达可以诱导肿瘤细胞的凋亡及细胞周期阻滞。

目的 构建人类乳头瘤病毒（HPV）16型晚期基因L1的原核表达质粒，以期从中选出能够高效表达HPV16L1的重组子，为进一步表达L1蛋白奠定基础。方法 应用定向克隆策略，将HPV16L1基因分别克隆到原核表达载体pTrac99A和pET -21c 中。结果　通过酶切鉴定选出了重组子pTrc99A -HPV16L1和pET-21c-HPV16L1。结论　HPV16L1的原核表达质粒构建成功。