HBV转录激活蛋白HBx、MHBst78MHBst155真核重组载体构建

目的分别构建人乙型肝炎病毒(HBV)X蛋白、羧基端截断的中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,以便进一步研究其转录激活功能及对宿主细胞信号传导通路的影响。方法设计合成3对寡核苷酸引物,以adr亚型HBV质粒pHBVDNA为模板,采用PCR法分别扩增HBVX基因、MHBst78与MHBst155编码基因片段;用HindⅢ,KpnⅠ双酶切HBVⅩ基因;用HindⅢ和BamHⅠ双酶切MHBst78与MHBst155编码基因片段后,分别定向插入到真核表达载体pcDNA3.1相应酶切位点,转化宿主菌JM109,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。结果酶切重组体显示所切下的片段大小均与预计相符,测序结果与文献报道序列及预计结果一致。结论成功构建了HBVX基因、羧基端截断的HBV中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,为进一步研究HBV转录激活蛋白HBx、MHBst78MHBst155对宿主细胞信号转导通路的影响奠定基础。     

同源框基因NKX3.1 mRNA和蛋白在前列腺组织特异性表达及与前列腺癌关系的探讨

目的:分析同源框基因NKX3.1 mRNA和蛋白在国人前列腺组织表达状况以及与前列腺癌关系。方法:采用半定量RT-PCR、蛋白杂交和免疫组化检测76例前列腺组织、96例非前列腺组织标本NKX3.1 mRNA和蛋白表达状况。结果:RT-PCR检测显示76例前列腺组织NKX3.1 mRNA表达率98.7%,96例非前列腺组织标本中,除2例睾丸组织、1例乳腺组织有NKX 3.1表达外,其余肾脏、膀胱、肝脏、回肠、脂肪、皮肤组织无1例表达(P<0.01)。蛋白杂交显示NKX 3.1蛋白总阳性表达率前列腺组织为100%,睾丸、乳腺组织均为16.7%,膀胱、回肠组织为8.3%,其他肾脏、肝脏、脂肪和皮肤组织均为0(P<0.01)。免疫组化检测显示NKX3.1蛋白主要分布在前列腺上皮细胞,上皮细胞总阳性表达率为94.7%,间质细胞NKX3.1蛋白总阳性表达率为5.3%;NKX3.1蛋白强阳性表达率良性前列腺组织为13.6%,前列腺癌组为40.6%(P<0.01)。结论:NKX3.1基因不仅为前列腺器官特异性基因,并且可能是前列腺腺上皮细胞特异性基因,其表达与前列腺癌关系密切。     

基因疫苗pcDNA-AChRα211免疫小鼠建立重症肌无力动物模型

目的：用基因疫苗pcDNA-AChRα211，免疫C57BL／6小鼠建立实验性自身免疫性重症肌无力模型（EAMG）。方法：将人乙酰胆碱受体α亚基N端的主要免疫区（AChRα211）的基因片段插入到穿梭载体pcDNA3．0中，构建基因疫苗pcDNA-AChRα211。大量提纯质粒pcDNA-AChRα211后肌肉注射C57BL／6小鼠。用ELISA法检测小鼠血清中抗AChRα211，的IgG（ACh-RAb），并用PCR方法检测外源基因在小鼠各组织器官中的分布情况。结果：双酶切鉴定和序列测定表明构建了含正确目的基因阅读框的重组质粒pcDNA-AChRα211，ELISA分析表明该基因疫苗免疫的C57BL／6小鼠血清中含有AChRAb，且免疫三个月后在小鼠的肌肉、肝、脾、肾中仍可检测到目的基因AChRα211的存在。结论：重组质粒pcDNA-AChRα211作为基因疫苗能够诱导实验性自身免疫性重症肌无力。     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

RAB5A正义表达载体对两种人肺腺癌细胞系粘附基底膜成份能力的影响

目的　RAB5A基因过表达与人肺腺癌细胞系Anip 973的高转移性相关 ,进一步确认该基因在人低转移肺腺癌细胞系SPC a1和人低分化肺腺癌细胞系GLC 82中的作用。方法　采用癌细胞粘附基底膜成分的测定方法 ,分析RAB5A正义真核表达载体转染后的SPC a1、GLC 82两种肺腺癌细胞系的侵袭能力的改变。结果　RAB5A正义表达载体PcDNA3 .1 RAB5A转染的SPC a1较未转染细胞与基底膜成分粘附能力明显增强 ,t检验P <0 0 1;GLC 82细胞的侵袭能力作用不明显。结论　RAB5A在侵袭转移表型形成中发挥一定的作用     

pcDNA3．1（＋）／NspA真核表达载体的构建及鉴定

目的构建pcDNA3.1( )淋病奈瑟菌外膜蛋白—奈瑟菌表面蛋白A(Neisseria surface Protein A,NspA)基因真核表达载体,为研制淋病奈瑟菌核酸疫苗奠定基础。方法根据淋病奈瑟菌NspA基因序列,设计合成一对引物,用PCR方法从淋病奈瑟菌基因组DNA中扩增出NspA基因片段,将扩增的产物连接于测序载体pUCm-T上,并构建重组体pcDNA3.1( )/NspA,进行酶切、PCR及测序鉴定。结果NspA基因体外扩增产物大小约为525 bp。所构建的pcDNA3.1( )/NspA重组体经双酶切及PCR鉴定,与预期片断的大小相符;测序结果与GenBank中收录的NspA全长序列一致,表明pcDNA3.1( )/NspA真核表达载体构建正确。结论成功地构建了淋病奈瑟菌真核重组表达载体pcD-NA3.1( )/NspA,为NspA蛋白的功能研究和淋病奈瑟菌核酸疫苗的研制提供了物质基础。     

转染survivin对HLF细胞增殖及凋亡的影响

 【【目的】 探讨survivin基因表达对人肺纤维母细胞（humanlungfibroblast，HLF）增殖、凋亡的影响。【方法】 构建survivin基因真核表达质粒peDNA3．1／SVN；利用脂质体转染法将其转入不表达survivin mRNA的HLF细胞系。连续培养10周。筛选出稳定表达的阳性克隆（HLF／SVN）及转空质粒对照（HLF／K）。用RT-PCR、免疫组化、Western blot检测细胞中survivin、bel-2和caspase3mRNA表达：通过观察细胞生长曲线、细胞凋亡指数。探讨转染survivin基因对HLF细胞生长、凋亡的影响。【结果】转染peDNA3．1／SVN后。在4、6、8和10周HLF／SVN细胞查见survivinmRNA表达；免疫组化和Westernblot见HLF／SVN细胞survivin阳性表达；转染后caspase3mRNA受到抑制，表达减弱或消失。HLF／SVN细胞凋亡指数（0．3％、1．0％、1．5％）皆低于HLF（0．9％、4．3％、2．1％）及HLF／K（0．8％、1．4％、2．8％）。细胞生长曲线示HLF／SVN细胞生长快于HLF和HLF／K，差异有统计学意义（P〈0．05）。【结论】survivin基因能促进HLF细胞生长。可能通过抑制凋亡和促进细胞分裂而发挥作用。     

Construction of eukaryotic expressing plasmids encodingHA andHA 1 of influenza A virus and their transient expression in HEK293 cells

Summary In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encodingHA andHA ₁ of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells.HA andHA ₁ genes were amplified by RT-PCR and cloned into pcDNA3. 1(+) to generate pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA₁, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA₁ were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA₁. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA₁, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine. Zhang Weidong, female, born in 1970, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 39970830).     

细胞内pH对胆碱能受体介导的Kir3.1/3.4电流的调节

目的　研究细胞内pH对胆碱能受体介导的Kir3 1/3 4电流的调节作用。方法　应用AzideNa、KHCO3 和通过灌流直接降低细胞内 pH ,用双电极电压钳和膜片钳方法观察在蛙卵细胞中表达的Kir3 1/ 3 4钾离子通道电流的变化和M受体激活对Kir3 1/ 3 4电流调节的变化。结果　细胞内 pH降低能抑制Kir3 1/ 3 4的电流 ;Kir3 1/ 3 4对细胞内pH的敏感性介于另外两种Kir通道Kir2 1和Kir2 3之间 ,即这三种通道对细胞内 pH的敏感性依次为Kir2 3>Kir3 1/ 3 4 >Kir2 1;细胞内pH降低能够减弱M1受体激活对Kir3 1/ 3 4的抑制作用 ,加强M2 受体激活Kir3 1/ 3 4电流后的去敏作用。结论　在维持细胞静息电位方面起重要作用的Kir3 1/ 3 4通道在细胞内pH降低的情况下基础电流和M受体激活调节电流均发生了变化 ,这些变化在缺血缺氧引起的细胞 (如心肌细胞 )兴奋性改变中可能有重要的生理学意义