人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.     

HIV-1Gag蛋白在大肠杆菌和重组杆状病毒系统中的高效表达及通用型温控原核表达载体的构建

将中国株HIV－1B亚型的gag全基因序列,克隆到杆状病毒表达载体pfastbacI中,构建了重组质粒pfastGag,利用细菌／杆状病毒表达系统筛选重组杆状病毒,在昆虫细胞中高效表达了HIV－1Gag蛋白。通过改造原核表达载体pBV220和pET28,构建了一种新的通用型温控原核表达载体质粒pVV5,该载体携带PrPl串联温控启功子及His—Tag纯化标签,利于目的蛋白表达与纯化。将HIV－1gag基因的1148一1857编码序列,分别插入到pVV5b、pET28b的相应位点,构建了重组表达质粒pEG1b、pEG7b,二者在不同受体菌中,表达重组蛋白的量分别占全菌体蛋白总量的42％和28％。利用IMAC金属螯合层析柱,对包涵体中的重组p24蛋白进行纯化,纯度超过80％;纯化后的重组蛋白可与HIV－1型标准阳性血清发生较强的免疫学反应。     

IBDV多聚蛋白基因真核表达质粒的构建与表达

将IBDV上海超强毒株的多聚蛋白基因(vp2-4-3)克隆入真核表达载体pALTER-MAX,构建成功pALTER-MAX-VP2-4-3真核表达质粒,经纯化后,pALTER-MAX-VP2-4-3在Lipofectamie^TM2000介导下转染Vero细胞、11日龄鸡胚的绒毛尿囊膜(CAM)和肌肉注射2日龄的雏鸡,1周后,分别提取细胞或组织中的总DNA或总RNA,用DIG标记探针均可检测到阳性杂交信号;转染的Vero细胞飞片和肌肉冰冻切片,进行免疫荧光检测均呈现阳性结果;转染的鸡胚CAM匀浆上清,用兔抗IBDV超强毒的高免血清,经Dot—ELISA检测呈现阳性。表明转染后基因获得表达,表达的蛋白具有免疫反应性。     

Expression in Bacillus subtilis of the glycosomal glyceraldehyde-phosphate dehydrogenase gene from Trypanosoma brucei.

The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.     

口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达

利用定点突变的原理,获得包含有口蹄疫病毒P1,2A,3C及部分2B编码区的目的基因片段,KpnⅠ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1（+）,经筛选、鉴定及DNA序列分析后,将重组质粒pcDNA3.1/P12X3C转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法,检测细胞中表达的口蹄疫病毒抗原。结果表明,口蹄疫病毒基因片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3.1/P12X3C可在BHK-21细胞中表达FMDV目的蛋白。     

B19病毒XA株VP1独特区真核表达系统的建立及鉴定

目的:克隆B19病毒XA株VP1u基因,构建真核重组表达载体.方法:从已构建好的B19病毒XA株原核表达载体中获得VP1u基因,将其克隆入真核表达载体plRES2-EGFP中,经酶切鉴定并测序验证后,获得真核表达载体plRES2-EGFP-VP1u.将其转染至HeLa细胞,提取细胞总蛋白,用Western blot技术检测VP1u蛋白的表达.结果:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u,荧光显微镜下可见pIRES2-EGFP-VP1u转染HeLa细胞后表达EGFP蛋白而发出绿色荧光,Western blot证明VP1u蛋白在HeLa细胞中表达.结论:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u并在HeLa细胞中正确表达,为今后B19病毒VP1u基因疫苗的研究奠定基础.     

HBV X基因的表达及在真核细胞中对内质网压力的作用

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上。重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后,IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清。重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Westernblot检测,证明HBx可以在这两种细胞系中表达。通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍。通过RT-PCR分析证明,转染了HBx的细胞中XBP1mRNA发生了剪切。因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础。     

含CSFV E2基因A/D区原核表达载体的构建、表达及其抗原性研究

应用RT PCR方法扩增了编码猪瘟病毒石门株 (CSFVshimenstrain)囊膜糖蛋白E2全基因 ,然后将其克隆到pMD 1 8T质粒中 ,获得重组质粒pMD E2。再以pMD E2为模板 ,另行设计两对引物 ,同时扩增其中一段适于在E .coli中表达且抗原反应性较好的基因片段 (E2蛋白A D抗原区基因序列 ) ,将扩增的两片段串联插入原核表达载体pET 32a中构建成重组质粒pET 2e。用酶切和序列分析鉴定插入目的基因的正确性。SDS PAGE和Western blot分析表明 ,经pET 2e转化、IPTG诱导的受体菌可表达目的蛋白 ,克隆在硫氧还蛋白 (thioredoxinprotein ,TrxA)基因下游的E2蛋白基因与TrxA基因获得了高效融合表达 ,并且具有免疫学反应活性 ,这为猪瘟的血清学诊断方法的建立打下了基础 。     

Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production

Corynebacterium glutamicum possesses phosphoenolpyruvate (PEP) carboxykinase, oxaloacetate decarboxylase and malic enzyme, all three in principle being able to catalyze the first step in gluconeogenesis. To investigate the role of PEP carboxykinase for growth and amino acid production, the respective pck gene was isolated, characterized and used for construction and analysis of mutants and overexpressing strains. Sequence analysis of the pck gene predicts a polypeptide of 610 amino acids showing up to 64% identity with ITP-/GTP-dependent PEP carboxykinases from other organisms. C. glutamicum cells harbouring pck on plasmid showed about tenfold higher specific PEP carboxykinase activities than the wildtype. Inactivation of the chromosomal pck gene led to the absence of PEP carboxykinase activity and the inability to grow on acetate or lactate indicating that the enzyme is essential for growth on these carbon sources and thus, for gluconeogenesis. The growth on glucose was not affected. Examination of glutamate production by the recombinant C. glutamicum strains revealed that the PEP carboxykinase-deficient mutant showed about fourfold higher, the pck-overexpressing strain two- to threefold lower glutamate production than the parental strain. Inactivation and overexpression of pck in a lysine-producer of C. glutamicum led to an only 20% higher and lower lysine accumulation, respectively. The results show that PEP carboxykinase activity in C. glutamicum is counteractive to the production of glutamate and lysine and indicate that the enzyme is an important target in the development of strains producing amino acids derived from citric acid cycle intermediates.     

荧光假单胞菌天冬氨酸转氨酶的基因克隆及其在大肠杆菌中的表达

采用克隆基因测序技术,从荧光假单胞菌GcM5-1A基因组文库中筛选到了天冬氨酸转氨酶的编码基因aspC。通过聚合酶链式反应（PCR）扩增目的基因,插入pET-15b构建重组表达质粒pET-15bAAT,转化E.coli BL21(DE3),IPTG诱导天冬氨酸转氨酶在大肠杆菌中高效表达,利用亲和层析法初步分离纯化了重组蛋白。生物活性分析表明,纯化的重组天门冬氨酸转氨酶具有氨基转移活性。     

重组虎源H5N1流感病毒HA基因犬2型腺病毒的构建与实验免疫研究

H5N1流感病毒可以对虎和猫产生致死性感染,为研制可用于预防猫科动物流感的新型疫苗,构建了重组虎源H5N1流感病毒HA基因的犬2型腺病毒。将A/Tiger/Harbin/01/2003(H5N1)的HA基因克隆入pVAX1载体中,然后将含有HA基因的表达盒(CMV HA PolyA)克隆入pVAXΔE3的SSPⅠ酶切缺失处,获得含有HA表达盒的穿梭载体pΔEHA。用SalⅠ NruⅠ分别对pΔEHA和pPoly-2-CAV2进行双酶切,将含有HA表达盒的SalⅠ NruⅠ片段克隆入pPoly2-CAV2,获得了在E3区缺失处插入HA表达盒的重组质粒pCAV-2/HA。释放CAV-2/HA重组基因组转染MDCK细胞,获得了重组活病毒CAV2/HA,经Western blot分析表明重组表达产物可被流感病毒HA单克隆抗体3A13所识别。使用该重组病毒免疫猫可以产生效价为1∶8~1∶16的抗H5亚型流感病毒血凝抑制抗体。     

人甲状旁腺激素(1-34)二联体与人血清白蛋白融合蛋白的构建及表达

构建人甲状旁腺激素(1-34)二联体与人血清白蛋白融合蛋白的表达载体,并表达得到该融合蛋白.通过设计强特异性的引物,利用重叠PCR技术,定向定量的拼接得到hPTH(1-34)二联体-HSA融合蛋白的基因;将构建好的融合基因插入表达载体pPIC9K,大量扩增重组质粒,并用Sal I线性化,电击转化毕赤酵母GS115,经组氨酸缺陷和G418抗性双重筛选得到阳性转化子;挑选阳性转化子进行甲醇诱导表达.测序结果表明得到的重组质粒pPIC9K-hPTH(1-34)二联体-HSA与目标设计完全一致;基因组PCR鉴定结果证明成功构建了hPTH(1-34)二联体-HSA融合基因的毕赤酵母(GS115)表达系统;SDS-PAGE电泳表明融合蛋白获得了表达,尿微量白蛋白试剂盒测定甲醇诱导表达3d后融合蛋白的产量为127 mg/L.     

人角质化细胞生长因子-2基因的克隆、表达及产物的纯化、鉴定

用高表达菌株BL21codon plus compentent cells表达重组人角质化细胞生长因子(Hkgf-2)蛋白并初步纯化和检测其活性。通过RTPCR从流产胎儿肺组织中钓取hKGF-2cDNA,将其克隆入pBV220载体质粒。在大肠杆菌BL-21codon plus compent cells中表达hKGF-2蛋白。采用亲和层析和离子交换层析分离纯化,以细胞增殖实验测定表达蛋白的生物活性。结果显示,hKGF-2蛋白在BL21中得到高效表达;hKGF-2蛋白能刺激NIH3T3细胞的增殖,具有显著的促有丝分裂活性。     

Phosphoenolpyruvate carboxykinase activity in grape berries

Phosphoenolpyruvate (PEP) carboxykinase activity was found in crude extracts of ;Pinot noir' grape berries. The enzyme required ATP, Mn(2+) plus Mg(2+), a pH of 6.6, and a temperature of 40 C for maximum activity. The range in concentration of oxaloacetic acid needed for maximum phosphoenolpyruvate carboxykinase activity was 5 to 10 mm, and the Km for HCO(3) (-) in the exchange of (14)CO(2) into oxaloacetic acid was 26.8 mm.Changes in the activity of PEP carboxykinase and PEP carboxylase in berries were studied at weekly intervals throughout fruit development. PEP carboxykinase had maximum activity 4 weeks after flowering, and during the following 11 weeks remained relatively constant. The activity of PEP carboxylase was 2- to 4-fold higher than PEP carboxykinase throughout fruit development, and changed little except for a sharp reduction at the onset of ripening.     

斑马鱼notch3基因真核表达载体的构建及其表达分析

为了进一步研究notch3基因在斑马鱼中的功能,构建了斑马鱼notch3真核表达载体并在真核细胞中成功表达.其中斑马鱼notch3基因编码序列(coding sequence,CDS)从NCBI的在线数据库中获得,根据序列克隆其胞内段(notch intracellular domain,NICD),接着利用同源重组技...     

癌胚抗原(CEA)启动子在肿瘤细胞中的特异表达及介异bak基因诱发凋亡

构建由癌胚抗原 (CEA)启动子控制报道基因增强型绿色荧光蛋白 (EGFP)表达的重组表达质粒pCEA EGFP .用转染细胞后检测荧光的方法对CEA阳性细胞进行简便、直观的检测 ,并结合流式细胞计数对CEA启动子在人结直肠腺癌细胞LS 1 74T、结肠癌细胞SW 4 80、肺腺癌细胞A5 49、人宫颈癌细胞HeLa和人喉癌细胞HEp 2中的活性进行了分析 ,发现其在SW4 80、LS 1 74T、A5 49中活性较强 ,而在HeLa和HEp 2中无活性 .构建由CEA启动子控制凋亡基因bak表达的重组表达质粒pCEA bak ,转染HeLa及SW 4 80细胞 ,用Hoechst332 5 8染色及PI染色 流式细胞计数分析的方法证明 ,pCEA bak转染能够特异性引起SW 4 80细胞的凋亡 .结果表明 ,CEA启动子具有很好的特异性 ,CEA介导bak基因的方法可望用于CEA阳性癌细胞的靶向性基因治疗 .     

多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达

目的：构建含有人存活蛋白（survivin）-2B主要T细胞表位区域、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域融合基因的真核表达质粒,并在人胚肾293T细胞中进行表达。方法：通过基因合成和搭桥PCR技术构建含有Survivin2B主要T细胞表位区域、人和猴CTP37区域基因的融合基因2PAG,将其插入含有人IgK链前导信号肽（sig）、人IgG-Fc和糖基磷脂酰肌醇（GPI）锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI—Fc—GPI中,继而又将酶切后的sig2PAG-FC-GPI融合基因导入含有细小病毒内部核糖体结合位点（IRES）基因且可以共表达人GM-CSF和B7．1融合基因的真核表达载体pVAX1-IRES-GM／B7中;将构建的重组质粒pVAX1-sig-2PAG-FC-GPI-GM／B7（简称pVAX1-2PFcGB）转染293T细胞,利用流式细胞仪和免疫荧光检测其表达情况。结果：2PAG融合基因经测序正确,PCR和酶切鉴定证明已成功连入真核表达载体pVAX-IRES-GM／B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-2PFcGB在293T细胞中得到很好的表达。结论：成功构建了重组质粒pVAX1-2PFcGB,且在293T细胞中可以有效表达,为对该基因疫苗的后续功能研究奠定了基础。     

牛α-IFN及FMDV P12A3C双表达载体的构建及其在BHK-21细胞中的表达

从口蹄疫病毒Asia I/Jiangsu毒株的细胞毒中提取总RNA,通过RT-PCR方法分别获得FMDV的P12A及3C基因;同时以pMD18-T-α-IFN质粒为模板,PCR扩增得到α-IFN基因.将α-IFN基因及FMDV P12A及3C基因连接至双启动子表达载体pBudCE4.1上,构建成双效表达质粒pBudCE4.1-α-IFN-P12A3C,经电泳、PCR、双酶切和DNA测序鉴定表明双效质粒载体构建成功.用此重组质粒转染BHK-21细胞后并对其表达情况进行检测,表明该双表达质粒在BHK-21细胞中能够成功表达.以此重组质粒免疫乳鼠后12 h,按100 TCID50/O.1 mL的量进行攻毒,结果发现该质粒能够抑制病毒的增殖,对乳鼠有一定的保护作用.结果表明成功构建了牛α-IFN及FMDV P12A3C组合基因双表达载体,为进一步研究口蹄疫基因疫苗提供前期基础.