重组人胰高血糖素样肽-1的表达、纯化及其生物学活性

为获得重组人胰高血糖素样肽 1[recombinanthumanglucagon likepeptide 1(7～ 37) ,rhGLP 1]并研究其生物学活性 ,采用亚磷酸二酯法合成hGLP 1cDNA的 6个寡核苷酸片段 ,拼接成完整的hGLP 1cDNA ,构建重组质粒pGEX hGLP 1,转化大肠杆菌BL2 1(DE3)获得表达菌株 .高密度发酵培养的菌体超声破碎后 ,裂解液用Glutathione Sepharose 4B亲和层析纯化得到GST融合蛋白 .经CNBr裂解、QAE SepharoseFF柱层析和脱盐 ,得到纯度大于 90 %的rhGLP 1,质谱测定分子量结果与理论值一致 .生物学活性分析表明 ,rhGLP 1具有明显的降血糖活性 .     

重组人促性腺激素释放激素及导肽的分离纯化与活性分析

对含重组人促性腺激素释放激素(GnRH)及导肽(LEP)的工程菌株进行诱导表达,分离纯化GnRH/LEP并进行生物学活性分析.工程菌IPTG诱导,收获的菌体经超声破碎后,裂解液用Glutathione-Sepharose 4B亲和层析纯化GST-GnRH/LEP融合蛋白,经CNBr裂解、Sephadex G-25柱脱盐、QAE-Sepharose FF阴离子交换柱层析和RP-C18柱脱盐,得到纯度大于98%的重组GnRH/LEP.Western blot表明表达产物均具有GrRH抗原特异性.生物学活性分析表明:该表达产物可促进小鼠次级卵泡向成熟卵泡发育,可提高其血清中E2含量.     

抗PAI抑制作用的纤溶酶原激活剂突变体的活性研究

目的:重组表达抗PAI抑制作用的t-PA突变体,经诱导表达、复性、纯化后进行生物学活性和酶动力学分析。方法:构建pBV220-tpa重组表达质粒,经DNA测序确认后,转化至大肠杆菌DH5a,温控诱导表达,凝胶过滤法对包涵体蛋白进行初步纯化,复性后,过刺桐胰蛋白酶亲和层析柱纯化,酶动力学分析其活性。结果:测序证实,t-PA突变体的DNA序列正确,表达蛋白占总菌体蛋白的30%,经纯化后纯度达90%以上,比活性为7.0×108IU/mg,t-PA突变体与PAI-1反应后,其活性未受到抑制。t-PA突变体酶的米氏常数Km为0.5298,最大水解速度Vmax为0.0595。结论:经生物学活性测定,表达蛋白能够明显抵抗PAI的抑制作用,并具有良好的生物活性,该突变体有可能成为用量更少、疗效更佳的新型溶栓药物。     

人白细胞介素18半胱氨酸的定点突变及其对活性的影响

为研究IL 18结构与功能的关系 ,用重叠延伸PCR定点突变技术构建人白细胞介素 18(hIL 18) 4个半胱氨酸的突变体hIL 18C74 S、C10 4 S、C112 S和C163 S。将突变体的cDNA与原核细胞表达载体pJW2重组并转化大肠杆菌JM10 1。经热诱导后 ,4个突变体在大肠杆菌中均得到了高效表达。表达的蛋白质主要以包涵体的形式存在。包涵体经超声破碎 ,2mol/L尿素洗涤 ,8mol/L尿素溶解 ,SephadexG 10 0柱纯化后 ,纯度可达 90 %以上。以诱导人外周血单个核细胞 (PBMC)产生IFN γ的能力为指标检测复性突变体的活性。结果显示除C10 4 S外 ,其他 3个突变体的生物活性均低于野生型hIL 18,C74 S、C112 S和C163 S的活性分别是野生型hIL 18活性的 5 %、5 8%和11%。证明Cys74 、Cys163 为hIL 18诱导产生IFN γ的功能所必需     

人凋亡相关基因TFAR19缺失突变体的原核表达、产物纯化及鉴定

构建TF 1细胞凋亡相关基因 19(TF 1cellapoptosisrelatedgene 19,TFAR19)缺失突变体的原核表达载体 ,获取缺失突变体蛋白 ,用于TFAR19促凋亡分子机理的研究 .从真核表达载体pcDI TFAR19扩增出野生型TFAR19和 4个缺失突变体 ,重组到原核表达载体pGEX 4T 2 .经亲和层析方法对缺失体蛋白进行纯化后 ,再利用凝胶过滤的方法进一步纯化 .利用抗GST和抗TFAR19的单克隆抗体对蛋白进行免疫学鉴定 .用白血病细胞株HL 6 0检测蛋白活性 .成功地克隆并重组了野生型TFAR19及缺失突变体 pGEX 4T 2表达载体 ,对融合蛋白的表达条件进行了优化 .SDS PAGE结果显示 ,各个缺失突变体融合蛋白均有较高水平的表达 .免疫学检测证实获得了正确的表达产物 .活性检测证实 ,野生型TFAR19和缺失突变体 4可以明显促进去血清诱导的HL 6 0细胞凋亡 ,第 6外显子可能是一个与TFAR19促凋亡活性密切相关的功能结构域     

甲醇酵母分泌表达重组人干扰素α1b纯化工艺的建立

建立适合大规模、低成本生产重组人干扰素α1b(Recombinanthumaninterferon α1b ,rhIFN α1b)的纯化工艺。采用高效分泌表达rhIFN α1b的甲醇酵母工程菌发酵 ,收集离心后的上清液 ,超滤脱盐 ,经离子交换柱和分子筛柱层析纯化。纯化的rhIFN α1b的纯度为 98%以上 ,比活性 2 .4× 10 7IU/mg ,活性回收率 14 % ,相对分子质量 1980 0和等电点 5 .0。经检测 ,rhIFN α1b蛋白N 端 15个氨基酸序列与正常对照完全符合。该纯化工艺简便 ,时程短 ,重复性好 ,适合于大规模生产     

人小肠三叶因子缺失半胱氨酸57突变体的构建及其生物活性

用 PCR方法构建了一个不能形成二聚体的 C端缺失半胱氨酸 57的 h ITF突变体 .将其克隆到大肠杆菌表达载体 p GEX- 4T- 1中 ,ITPG诱导表达 ,融合蛋白经 Glutathione- Sepharose 4B亲和层析 ,凝血酶酶切和 Sephacryl S 1 0 0纯化 ,得到突变体蛋白 .SDS- PAGE,氨基酸组成 ,飞行质谱 ,N端氨基酸序列测定结果与期望值一致 .研究表明突变体的生物学活性有所降低 .对胃蛋白酶作用的稳定性降低 .     

低出血倾向、低免疫原性葡激酶mSAK(K130T,K135R)的表达、纯化与活性测定

为了有效降低葡激酶应用的副作用,构建低出血倾向、低免疫原性葡激酶突变体,高效表达纯化后进行活性鉴定,以野生型重组葡激酶基因为模板,PCR法引入突变位点(K130T,K135R),并将该片段克隆测序鉴定后,克隆至表达载体pBV220,构建低免疫原性葡激酶突变体.表达后的蛋白用Q-SepharoseFF柱与SephacrylS-200进行纯化,纤维蛋白溶圈法进行活性测定,体外抗体中和试验与豚鼠免疫试验测定突变体蛋白的免疫原性,同时进行动物体内出血倾向观察.测序结果表明,相应位点获得突变,无非特异性突变,将突变后的片段连接pBV220导入大肠杆菌热激诱导获得了高效表达,表达产物占菌体总蛋白的40%~50%.产物主要以可溶性形式存在,经两步纯化后的蛋白的纯度可达98%以上.活性测定试验表明,该突变体的活性较野生型葡激酶稍低,体外中和抗体试验和豚鼠免疫试验证明免疫原性大为下降,豚鼠的皮肤出血以及肺部病理切片均显示该突变体蛋白引起的出血倾向明显降低.     

Asp^126、Asp^130、Asp^134对人白介素18功能的影响

为研究人白细胞介素18(hIL-18)中一些氨基酸残基对IL－18功能的影响,用重叠延伸PCR定点诱变技术构建hIL-18突变体hIL-18D^126N、hIL-18D^130K、hIL-18D^134K。将突变体cDNA与原核表达载体pJW2重组并转化大肠杆菌DH5α。经热诱导表达,3个突变体占菌体总蛋白质的15％－31％以上。SDS－PAGE证实,表达的蛋白质以包含体形式存在。包含体经超声破坏,2mol/L尿素洗涤,8mol/L尿素溶解,Sephadex G-75柱纯化后,纯度均可达95％以上。Western印迹表明3个突变体与野生型hIL-8具有相同的免疫原性。纯化的突变体蛋白质经复性后,以诱导人外周血单个核细胞（PBMC）产生于干扰素（IFN－γ）的能力为指标检测其活性,结果显示3个突变体的生物学活性分别为野生型IL－18的32％、8％、10％,表明hIL-18中126、130、134位的天冬氨酸（Asp)对其功能是必需的。     

单纯疱疹病毒1型囊膜糖蛋白gD在大肠杆菌中的表达、纯化及其免疫活性的鉴定

目的:在大肠杆菌中表达1型单纯疱疹病毒(HSV-1)囊膜糖蛋白gD,纯化重组蛋白并对其免疫活性进行鉴定。方法:将HSV-1 gD 基因克隆入原核表达载体pET-28b,利用异丙基-B-D-硫代吡喃半乳糖苷(IPTG)诱导重组质粒转化的大肠杆菌,探讨IPTG浓度、诱导时间、诱导温度对重组蛋白表达的影响;盐酸胍裂解变性包涵体,镍柱亲和层析法纯化gD蛋白,并对纯化后的蛋白进行透析复性;Western blot和ELISA检测gD蛋白的免疫活性。结果:酶切和测序结果表明gD基因克隆入pET-28b载体。该重组质粒转化的大肠杆菌经IPTG诱导后重组蛋白主要以包涵体形式存在,大小约40kDa。gD蛋白诱导表达的最佳条件为0.5mmol/L IPTG于37℃诱导8h。镍柱亲和层析法纯化获得的gD蛋白总量为3.1mg/L,透析复性后获得的gD蛋白总量为1.3mg/L,复性率为41.37%。Western blot及ELISA检测表明表达的gD蛋白具有免疫活性。结论:在大肠杆菌中表达并纯化获得具有免疫活性的HSV-1 gD蛋白,为进一步制备HSV-1诊断试剂和预防疫苗奠定了基础。     

人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达

目的：在巴斯德毕赤酵母中表达有降糖活性的人胰高血糖素样肽-1（hGLP-1）突变体（2Gly-hGLP-1）与人血清白蛋白（HSA）的融合蛋白。方法：为将GLP-1氨基酸序列第2位的丙氨酸（Ala）定点突变为甘氨酸（Gly）,根据毕赤酵母偏爱密码子合成编码2Gly-hGLP-1的基因;采用重叠PCR法拼接2Gly-hGLP-1和HSA的基因,使得2Gly-hGLP-1的C端与HSA的N端通过甘氨酸五肽接头连接;将该融合基因插入表达载体pPIC9构建为重组载体pPIC9/2Gly-hGLP-1-HSA,电击转化至毕赤酵母GS115细胞,通过表型筛选和诱导表达实验获得高效表达菌株;工程菌在5L发酵罐中培养后,对发酵产物进行分离纯化和生物学活性分析。结果：融合蛋白在5L发酵罐中的表达量约为200mg/L,经纯化后纯度可达95%以上;小鼠糖耐量实验表明该融合蛋白具有明显的控血糖活性。结论：在毕赤酵母中分泌表达的融合蛋白2Gly-hGLP-1-HSA具有降血糖活性。     

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.     

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.     

一种白细胞介素2基因变构体的高效表达与纯化工艺

利用定点诱变技术构建表达质粒pET15b-MhIL-2并将其在大肠杆菌中进行表达发酵的优化研究,高效表达出可溶性的MhIL-2重组蛋白。蛋白经过亲和层析、Thrombin酶切、离子交换层析和凝胶过滤层析纯化,MhIL-2纯度达95%,且MhIL-2比hIL-2具有更强的促进T细胞增殖生物活性。     

Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation.

Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (F0) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein.     

重组人碱性成纤维细胞生长因子(bFGF)融合蛋白的高效表达及鉴定

碱性成纤维细胞生长因子（ｂＦＧＦ）参与了许多细胞生长和分化的调控过程。本文采用重组ＤＮＡ技术在大肠杆菌中高效表达了人ｂＦＧＦ。首先将编码人ｂＦＧＦ基因克隆到ｐＸＴ表达载体中与其上游的一短Ｓ导肽共一阅读框架,ｂＦＧＦ基因的表达受强的Ｔ７启动子调控。采用ＢＬ２１（ＤＥ３）大肠杆菌作为宿主菌,用ＩＰＴＧ诱导ＢＬ２１（ＤＥ３）细菌合成的Ｔ７ＲＮＡ聚合酶,后者可催化高水平的ｂＦＧＦ基因表达,其ｂＦＧＦ产量可占总菌体蛋白的４２.５％。采用肝素Ｓｅｐｈａｒｏｓｅ一步亲和层析法直接从诱导后的细菌裂解产物中得到纯化的重组人ｂＦＧＦ蛋白。经Ｗｅｓｔｅｒｎ印迹分析证明该蛋白可被人ｂＦＧＦ特异性单克隆抗体所识别。进一步研究证明该蛋白具有刺激ＮＲ６Ｒ－３Ｔ３成纤维细胞增殖的生物学活性,并且这一活性可被人ｂＦＧＦ特异性中和抗体所中和。     

重组人胰高血糖素样肽－1的表达及生物学活性

将人工合成的人胰高血糖素样肽-1（human glucagon like peptide-1, hGLP-1）基因插入质粒载体pET-32a(+)中,构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达载体pET32-GLP-1,转化大肠杆菌BL21（DE3）获得表达菌株,经IPTG诱导发酵的菌体超声破碎后,裂解液用Ni离子亲和层析纯化得到融合蛋白,经肠激酶裂解,再次Ni离子亲和层析,得到rhGLP-1样品。经SDS PAGE 和等电聚焦检测,样品纯度大于90％, 等电点介于pH5.2～pH5.85之间。质谱测定rhGLP-1分子量为3 355.0kDa,肽图分析得到2 097.7kDa和1 005.5kDa两个胰蛋白酶酶解片断,均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。