人补体调节蛋白DAF、MCP在哺乳动物细胞中的共表达及协同作用研究

利用双启动子构建含人补体调节蛋白DAF和MCP cDNA的双顺反子重组表达载体pcDNA3-DAFMCP-DP, 以磷酸钙沉淀法转染NIH3T3细胞, 用G418筛选获得NIH3T3 pcDNA3-DAFMCP-DP转化细胞。PCR实验结果显示人补体调节蛋白基因DAF和MCP整合在转化的异源细胞的染色体上。RT-PCR和Western blot印迹实验分别从RNA水平和蛋白质水平证实了人补体调节蛋白分子DAF和MCP在细胞系中皆获得同步表达。检测连续传代30次的NIH3T3 pcDNA3-DAFMCP-DP结果表明人DAF和MCP基因仍稳定整合在细胞基因组中, 并未随着传代而丢失, 为稳定的转双基因细胞系。补体依赖的细胞毒反应表明, pcDNA3-DAFMCP-DP转染细胞系由于DAF和MCP的共表达获得较DAF或MCP单一表达时更强的保护能力, 能更好地抑制人补体依赖的细胞毒作用的发生, 保护宿主细胞免受人补体的攻击。以上结果表明, DAF和MCP双基因重组表达载体实现了人补体调节蛋白基因高效转移和高水平共表达, 为获得表达多种人补体调节蛋白的理想供体提供了有效策略。而且共表达的DAF和MCP具有协同效应, 能更有效地阻止补体激活造成的细胞损伤, 在克服超急性排斥反应的基因治疗中具有潜在的临床应用价值。     

OTX1基因慢病毒载体的构建及过表达研究

OTX1基因是神经发育调控中关键转录因子之一。本实验构建表达OTX1基因慢病毒载体,探讨慢病毒介导OTX1基因体外过表达的可行性。将OTX1基因克隆到慢病毒穿梭质粒DUET101内,构建表达OTX1以及GFP－OTX1基因的慢病毒载体;将pDUET－OTX1、pDUET－GFP－OTX1及pDUET101（空载体对照）质粒分别与慢病毒包装质粒pCMV R8.91、pMD.G共同转染人胚胎肾上皮细胞系293T细胞,获得携带OTX1基因、GFP－OTX1基因的重组慢病毒DUET－OTX1、DUET－GFP－OTX1以及仅携带GFP基因的DUET－GFP;用重组慢病毒分别转导293T细胞、SY5Y细胞、小鼠胚胎干细胞及大鼠胚胎15天皮层神经干细胞,证实慢病毒载体能够将外源基因转导入不同细胞,且转导的OTX1或GFP－OTX1在不同细胞中均仅在细胞核内表达;培养OTX1单克隆抗体细胞,浓缩抗体上清,通过Western Blot以及细胞免疫荧光法证实慢病毒介导的OTX1基因及GFP－OTX1基因转导入293T细胞后的过表达。本实验证实慢病毒载体是高效的基因转移载体;OTX1作为发育过程中至关重要的转录因子,经过重组慢病毒体外转导后均只在细胞核内表达。     

人源抗TNF—α抗体Fab基因的单链化及其表达和纯化

构建人源抗TNF－α单链抗体基因,并尝试其在E.coli中的表达和纯化,采用人工接头,按VH－linkerVL的结构将人源抗TNF－a的VH,VL基因拼接成单链抗体（ScFv)基因,并将构建好的ScFv基因插入表达载体pQE30,转染E.coli M15,以IPTG诱导表达,Ni-NTA树脂纯化表达产物,构建的ScFv基因长723bp, 列分析表明,该序列拼接正确,SDS－PAGE显示,用重组的pQE30转化的M15菌经诱导后,有相对分子质量（M）约为52000的外源蛋白表达,Ni-NTA树脂纯化的表达产物纯度大于90％,因此,成功地构建了人源抗TNFa的ScFv基因,并在E.coli DH5a中表达和纯化的该基因的产物。     

重组人GM—CSF基因在昆虫细胞中的表达

利用苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标记的非融合蛋白基因转移载体ｐＢｌｕｅＢａｃ将人粒细胞巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）基因成功地插入病毒ＡｃＮＰＶ的基因组中．ｈＧＭ－ＣＳＦ基因在感染重组病毒的草地夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）培养细胞Ｓｆ９中得到表达,感染后的Ｓｆ９细胞培养液能刺激人骨髓细胞在体外形成典型的集落,表达水平可达２．７×１０５５ＣＦＵ／ｍｌ。以ｈＧＭ－ＣＳＦ单抗所作的ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ表明,表达的ｈＧＭ－ＣＳＦ对是３种糖基化程度不同的产物,分子量分别约为１５ｋｄ,１８ｋｄ和２０ｋｄ。     

失活 ChiA 和 v-cath 基因的重组BmNPV 表达 hGM-CSF

利用 DNA 同源重组技术使家蚕核型多角体病毒的多角体蛋白基因取代 v-cath 、 ChiA 两基因的部分编码区域和共同的启动子区域,获得了 v-cath 、 ChiA 两基因同时失活的、可形成多角体的、并能表达人粒细胞 - 巨噬细胞集落刺激因子的重组病毒 BmNPVpolh+CP－ChiA－GMCSF+. 研究结果显示： v-cath 、 ChiA 两基因的失活不影响病毒的复制及多角体的形成,但感染细胞的存活时间比野生病毒感染的多 2 天,可明显改善外源基因在培养细胞和家蚕中的表达水平;感染 BmNPV polh+CP－ChiA－GMCSF+的家蚕幼虫体表保持正常,无野生病毒感染后引起的破皮流脓现象 .     

表达粒细胞巨噬细胞集落刺激因子的非复制痘苗病毒的抗肿瘤效果

构建了表达GM－CSF的非复制型重组痘苗病毒RV△11β75GMCSF。Southern blot证实重组病毒C－K片段间基因缺失,同时插入GM－CSF 基因,GM－CSF可以分泌形式良好表达。Wistar大鼠Walker‘s瘤内注射重组病毒RV△11β75GMCSF后,可以延长荷瘤鼠的生存时间,重组病毒RV△11β75GMCSF修饰的肿瘤细胞裂解物（WRC256）的肿瘤免疫治疗,可以抑制荷瘤大鼠的肿瘤生长,瘤重较对照组减轻,此结果提示,非复制重组痘苗病毒表达的GM－CSF可作为有效的肿瘤免疫治疗佐剂。     

含人TNF-α、IL-2及NeoR基因多顺反子逆转录病毒载体的构建及表达

利用脑炎心肌炎病毒和脊髓灰质炎病毒的内核糖体进入位点,连接人ＴＮＦ－α及ＩＬ－２ｃＤＮＡ和选择基因新霉素磷酸转移酶基因（ＮｅｏＲ）,使ＴＮＦ－α、ＩＬ－２及ＮｅｏＲ基因均受控于病毒ＬＴＲ启动子,将这３个基因转录至同一ｍＲＮＡ,从而构建成含人ＴＮＦ－α、ＩＬ－２及ＮｅｏＲ基因多顺反子逆转录病毒载体ｐＧＣＥＮ／ＴＲＩ。在ＬｉｐｏｆｅｃｔＡＭＩＮＥ介导下将其导入包装细胞ＰＡ３１７,Ｇ４１８筛选得单克隆,病毒滴度为５×１０５ＣＦＵ／ｍｌ重组病毒分泌的细胞株,经ＰＣＲ证明外源基因已整合至基因组,Ｎｏｒｔｈｅｒｎ印迹显示出单一转录本。用重组病毒上清感染不同的细胞,Ｇ４１８筛选所获得的抗性克隆能不同程度地表达ＴＮＦ－α及ＩＬ－２。实验结果表明,含ＩＲＥＳ的多顺反子逆转录病毒载体能很好地在同一靶细胞中表达多个外源基因。     

人TNF-α双顺反子逆转录病毒载体的构建及其在成纤维细胞中的表达

利用脑炎心肌炎病毒的内核糖体进入位点连接人ＴＮＦ－αｃＤＮＡ和选择基因ＮｅｏＲ基因,使ＴＮＦ－α及ＮｅｏＲ基因均受控于病毒ＬＴＲ启动子,将两基因同时转录至同一ｍＲＮＡ,从而构建成人ＴＮＦ－α双顺反子逆转录病毒载体ｐＧＣＥＮ／ＴＮＦ－α．在ＬｉｐｏｆｅｃｔＡＭＩＮＥ介导下将其导入包装细胞ＰＡ３１７,Ｇ４１８筛选得单克隆,病毒滴度为１０６ＣＦＵ／ｍｌ重组病毒分泌的细胞株．经ＰＣＲ证明外源基因已整合至细胞基因组,Ｎｏｒｔｈｅｒｎ印迹显示出单一ＬＲＴ转录本．持续Ｇ４１８筛选能明显促进目的基因ＴＮＦ－α的表达．用重组病毒上清感染小鼠成纤维细胞ＮＩＨ３Ｔ３,Ｇ４１８筛选获得的混合抗性克隆持续高表达ＴＮＦ－α,４０Ｇｙγ线照射后能维持高效表达至７ｄ．实验结果表明,含ＩＲＥＳ的双顺反子逆转录病毒载体将是一个很好的基因转移载体．     

异种器官移植用血管内皮细胞特异表达人CD59重组基因的构建

PCR插入法将人细胞间粘附分子（ICAM－2）启动子、人CD59－enhancer克隆到human CD59cDNA-pcDNA3表达质粒中,进行序列测定及分析。成功地构建了异种器官移植用血管内皮细胞特异表达人CD59重组基因,提供了CD59重组基因用于转基因动物的研究。     

一种新型人肿瘤坏死因子突变体在大肠杆菌中的高效表达

采用多位点突变引物和ＰＣＲ方法对合成的人肿瘤坏死因子进行了突变,通过这一突变,人肿瘤坏死因子的第二位精氨酸的密码子ＣＧＴ被变为赖氨酸密码子ＡＡＡ,将突变后的基因插入表达载体ｐＳＢ－９２的ＰＬ启动子下游得到重组质粒ｐＳＢ－ＴＮＦ－Ｋ２,并在大肠杆菌中进行表达。突变体［Ｌｙｓ２］ｈＴＮＦ－α的表达产率达到菌体内总蛋白质的６０％以上,且为一可溶性蛋白质并易于纯化。同野生型ｈＴＮＦ－α相比,［Ｌｙｓ２］ｈＴＮＦ－α具有稍低的毒性,但对于某些人肿瘤细胞株表现出高于数十到数百倍的抑制活性．     

Additional forms of human decay-accelerating factor (DAF)

Decay-accelerating factor (DAF) of human erythrocytes is a glycoprotein with a Mr of 65,000 that is anchored in the membrane via a glycolipid tail. During the purification of DAF, two lower m.w. forms were noted. DAF-A had an Mr of 63,000, and DAF-B had an Mr of 55,000. In a fluid phase assay, both forms accelerated the decay of the classical and the alternative C3 convertases with a specific activity similar to that of DAF. However, the decay-accelerating activity for the cell-bound C3 convertases was abolished, suggesting that neither could insert into E membranes and therefore that the glycolipid tail is altered. Analysis by molecular sieve high-pressure liquid chromatography demonstrated that DAF-A eluted with a Mr of approximately 450,000, similar to native DAF, and was thus in an aggregated form. In contrast, DAF-B eluted as a monomer with a Mr of approximately 60,000. DAF-A, but not DAF-B, bound to a hydrophobic column. To further characterize these two forms, surface-labeled human erythrocytes were incubated with phosphatidyl inositol-specific phospholipase C or papain. The phospholipase inefficiently released a form of DAF that was slightly larger (Mr of 64,000) than DAF-A. Papain efficiently released a 55,000 fragment that had the same Mr as DAF-B. To determine if DAF was cleaved by endogenous enzymes, surface-labeled erythrocytes were incubated with leukocytes. The kinetics of the leukocyte-induced degradation was similar to those observed with papain, and the released fragment aligned on seizing gels with the papain-derived fragment. We hypothesize that endogenous phospholipases and proteases cleave DAF to produce fragments similar to DAF-A and DAF-B, respectively.     

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.     

Cooperation between human DAF and CD59 in protecting cells from human complement-mediated lysis

The complement (C) regulatory proteins decay accelerating factor (DAF, CD55) and CD59 could protect host cells using different mechanisms from C-mediated damage at two distinct levels within the C pathway. Co-expression of DAF and CD59 would be an effective strategy to help overcome host C-induced xenograft hyperacute rejection. In this study, we made a construct of recombinant expression vector containing DAF and CD59 cDNA and the stable cell lines were obtained by G418 selection. Extraneous genes integration and co-expression were identified by PCR, RT-PCR and Western blot analysis. Human c-mediated cytolysis assays showed that NIH/3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-DAF, and pcDNA3-CD59DAF-DP were protected from Cmediated damage and that synchronously expressed human CD59 and DAF provided the most excellent protection for host cells as compared with either human CD59 or DAF expressed alone. Therefore, the construct represents an effective and efficacy strategy to overcome C-mediated damage in cells and, ultimately, in animals.     

The isolation,characterisation, and chromosomal assignment of the gene for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, (HMG-CoA reductase)

Summary We have used a cDNA clone for Chinese hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to isolate a genomic recombinant for human HMG-CoA reductase. The identity of the gene was confirmed by partial sequence analysis. Several unique fragments that will be useful for restriction fragment length polymorphism (RFLP) studies were identified. In situ hybridisation of a 2.6kb unique fragment of the gene to human metaphase chromosomes localised the human HMGCoA reductase gene to human chromosome 5q12.     

Decay-accelerating factor functions as a signal transducing molecule for human monocytes.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that protects cells from damage by autologous complement activation. Of the four mAb against DAF prepared in our laboratory, 1C6 completely blocked DAF function, whereas 5B2 partially blocked it. Using these mAb, we investigated whether human monocytes were activated via DAF molecules. When monocytes were incubated with 1C6 alone, glucose was consumed in significant amounts and phagocytosis of latex beads was enhanced, indicating that the monocytes had been activated. However, 1C6 did not enhance the production of monokines, TNF-alpha, and IL-1 alpha and -beta. The F(ab')2 fragment of 1C6 also activated monocytes, whereas 5B2 and the Fab fragment of 1C6 could not. To further examine monocyte activation, these cells were treated with phosphatidylinositol-specific phospholipase C. Increased glucose consumption and enhanced phagocytic activity by 1C6 were considerably reduced in monocytes treated with phosphatidylinositol-specific phospholipase C. In addition, we found that 1C6 stimulated the generation of inositol trisphosphate. These results demonstrate that the signal transmitted via the DAF molecule is capable of stimulating monocytes.     

人成骨蛋白成熟肽基因在毕赤酵母中的克隆及其序列分析

采用PCR方法,根据献报道的人成骨蛋白-1（OP-1）成熟肽基因序列,设计并合一对引物,从含人成骨蛋白基因的质粒中扩增获得大小的420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,继之以pPIC3.5k为表达载体构建重组表达质粒,并经PCR及酶切鉴定。     

人核糖核酸酶抑制因子基因重组腺病毒载体的构建及鉴定

目的构建含有人核糖核酸酶抑制因子（hRI）基因的重组腺病毒载体。方法以含有全长cDNA的pT7-RI为模板,PCR扩增hRI,经T载体克隆后,酶切亚克隆到穿梭质粒pAdTrack—CMV上,在BJ5183细菌内和pAdEasy-1同源重组。筛选阳性克隆,酶切、PCR及测序鉴定,线性化后脂质体法转染293细胞进行包装、扩增。通过观察绿色荧光蛋白（GFP）的表达及PCR扩增目的基因等方法鉴定重组的腺病毒。结果酶切鉴定及PCR结果证明hRI基因重组腺病毒载体构建成功,病毒滴度为1．5×10^10 pfu/ml。结论应用细菌内同源重组法成功构建了含hRI基因的重组腺病毒载体。     

牛SRY同源基因的PCR扩增和克隆

本文采用人SRY基因的一对引物,通过PCR扩增获得了雄性牛(Bos taurus)SRY同源基因片段。进一步证实牛存在与人SRY基因同源的相应基因。将PCR产物与载体pUC—Eco—T连接后,用以转化JM109菌,经过与人SRY基因探针菌落杂交,筛选获得了牛SRY同源基因克隆pBosY O.6后者的插入片段为相应于人SRY基因保守区在内的一段约609bp DNA。此外还比较分析了人和牛SRY同源基因片段限制酶图谱。     

APC11基因真核表达质粒构建及鉴定

目的克隆扩增APC11开放阅读框基因片段,构建其真核表达重组质粒,为后续研究其功能做准备。方法按照GenBank中人APC11序列设计引物,以HeLa细胞cDNA为模板,扩增出基因片段。该片段与真核表达载体pEGFP-C1连接,得到的重组质粒经双酶切和测序鉴定后用Western印迹检验表达。结果扩增片段长度为285bp,重组真核表达质粒经双酶切鉴定后测序鉴定正确。Western印迹证实pEGFP—C1-APC11在真核细胞内表达。结论成功克隆了APC11基因并构建了pEGFP—C1-APC11真核表达重组质粒,Western印迹证实其表达良好,对该基因功能的深入研究打下了坚实基础。