苏云金芽胞杆菌Sigma K在大肠杆菌中的表达与纯化

【目的】在大肠杆菌中表达纯化苏云金芽胞杆菌HD73的转录调控因子Sigma K(σK)。【方法】PCR扩增出苏云金芽胞杆菌HD73中sig K基因的ORF(Open reading frame)装载到带有His标签的表达载体p ET21b上,转入到表达菌株BL21(DE3)中获得重组菌株BL21(p ETsig K),通过SDS-PAGE、镍柱亲和纯化、阴离子交换纯化和凝胶迁移实验(EMSA)等方法对Sigma K蛋白进行提取、纯化和生物活性分析。【结果】正确表达出大小约为27 k D的His-Sigma K蛋白,并获得了纯化的蛋白。EMSA结果表明纯化的His-Sigma K蛋白可以与受其控制的cry1Ac基因启动子结合。【结论】表达和纯化了His-Sigma K蛋白,His-Sigma K具有与受其控制的启动子结合的功能。     

PTD融合蛋白的原核表达及其抗体制备

目的：为制备凋亡素重组蛋白抗体,首先获得凋亡素重组蛋白融合基因LTA,而且通过原恢表达系统表达重组蛋白并制备其抗体,为进一步利用凋亡素重组蛋白导向治疗肿瘤的检测奠定基础。方法：应用重叠延伸的基因融合技术将LHRH（黄体生成激素释放激素）基因、TAT（HW—1反式转录激活因子）基因和凋亡素基因重组,构建成凋亡素重组蛋白原核表达载体pET-28α～LTA,随后将表达质粒转入BL21菌株,经IPTG诱导表达重组融合蛋白,将已表达的重组蛋白通过Ni—NTA亲和色谱柱进行纯化,并制备LTA凋亡素重组蛋白抗体。结果：表达产物经聚丙烯酰胺凝胶电泳检测．LTA蛋白融合基因获得高效表达,凝胶薄层扫描分析表明表达蛋白占菌体蛋白12．6％。LTA蛋白经Ni—NTA亲和色谱柱柱纯化,以纯化蛋白为抗原免疫獭兔制备凋亡素重组蛋白抗血清。结果表明抗体的效价为1：12800。结论：应用重叠延伸的基因融合技术获得凋亡素重组蛋白融合基因LTA,通过原核表达系统表达重组蛋白并制备其抗体。     

人肝再生增强因子在毕赤酵母中的表达、纯化和生物学活性的研究

肝再生增强因子（ALR）是一类胞源性肝细胞生长因子。为在毕赤酵母中分泌表达人肝再生增强因子（rhALR）,以色谱法分离纯化后进行体外活性研究,构建表达载体pPICZαA- ALR,经电穿孔转入毕赤酵母中,用0.5％甲醇诱导表达;重组酵母培养上清经SDS-PAGE电泳和western blot鉴定后表明, rhALR以分子量为30kD的二聚体为主;定量分析结果表明,重组酵母培养上清中rhALR约占总蛋白的66％,表达量约为40mg/L;经DEAE柱和G75柱纯化后,获得的rhALR纯度大于95％,得率为52％;体外生物学活性实验表明,rhALR能明显促进HepG2、SMMC-7721和NIH-3T3细胞的增殖。     

血红蛋白基因在产CBHⅡ毕赤酵母工程菌中的表达

目的:提高毕赤酵母工程菌P.pastoris-CBHⅡ液体发酵产纤维二糖水解酶(CBHⅡ)的能力。方法:分别构建了用于胞外及胞内表达透明颤菌血红蛋白(VHb)基因的毕赤酵母重组表达质粒pPICZαA-vhb与pPICZαA(-s)-vhb。通过电转化将两种质粒转化巴氏毕赤酵母重组菌体P.pastoris-CBHⅡ菌株,经过筛选分别获得可正确表达具有生物活性的VHb蛋白的重组菌株P.pastoris-CBHⅡ-vhb(胞内表达VHb)及P.pastoris-CBHⅡ-vhb(-s)(胞外表达VHb)。结果:摇瓶发酵实验表明,血红蛋白在贫氧条件下可促进CBHⅡ的分泌表达,培养液上清的CMC酶活分别从出发菌株P.pastoris-CBHⅡ的1.81U/ml提高到2.04U/ml(胞内表达VHb)与1.93U/ml(胞外表达VHb)。VHb蛋白胞内表达菌株比胞外表达菌株效果更明显。     

PTD融和蛋白的原核表达及其抗体制备

目的:为制备凋亡素重组蛋白抗体,首先获得凋亡素重组蛋白融合基因LTA,而且通过原核表达系统表达重组蛋白并制备其抗体,为进一步利用凋亡素重组蛋白导向治疗肿瘤的检测奠定基础.方法:应用重叠延伸的基因融合技术将LHRH(黄体生成激素释放激素)基因、TAT(HIV-1反式转录激活因子)基因和凋亡素基因重组,构建成凋亡素重组蛋白原核表达载体pET-28a-LTA,随后将表达质粒转入BL21菌株,经IPTG诱导表达重组融合蛋白,将已表达的重组蛋白通过Ni-NTA亲和色谱柱进行纯化,并制备LTA凋亡素重组蛋白抗体.结果:表达产物经聚丙烯酰胺凝胶电泳检测,LTA蛋白融合基因获得高效表达,凝胶薄层扫描分析表明表达蛋白占菌体蛋白12.6%.LTA蛋白经Ni-NTA亲和色谱柱柱纯化,以纯化蛋白为抗原免疫獭兔制备凋亡素重组蛋白抗血清.结果表明抗体的效价为1: 12800.结论:应用重叠延伸的基因融合技术获得凋亡素重组蛋白融合基因LTA,通过原核表达系统表达重组蛋白并制备其抗体.     

狂犬病病毒重组免疫毒素的表达及其生物活性鉴定

将狂犬病病毒中和性单链抗体基因克隆入原核表达载体pET-PE40,经酶切鉴定及序列测定,成功构建了重组免疫毒素原核表达载体。IPTG诱导后目的蛋白获得高效表达,SDS-PAGE分析目的蛋白主要以不溶性包涵体的形式存在于菌体中,表达量占菌体总蛋白的32.29％。包涵体蛋白经体外复性及离子交换色谱柱、疏水作用色谱柱、Sephadex G200凝胶过滤层析柱三步纯化后获得纯度大于96％的目的蛋白,间接免疫荧光染色检测表明重组免疫毒素与狂犬病病毒感染细胞具有抗原结合活性,MTT试验显示,重组免疫毒素对狂犬病病毒感染细胞具有明显的杀伤作用,而对正常细胞无杀伤作用。     

HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

为研究乙肝核心抗原蛋白(HBcAg)在甲醇型酵母(PichiaPastoris)中的表达和性质,用PCR方法将HBcAg基因(L)克隆到P.Pastoris胞内表达载体pPIC3k中,并利用电击和同源重组法,将重组质粒pPIC3kL转化感受态的甲醇型GS115酵母菌株。经过筛选得到阳性P.Pastoris重组子。重组菌株经甲醇诱导培养,表达产物的Western印迹结果表明,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达,产物为一215kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为12576gml和13013gml的2个峰值处。电镜观察表明,该重组HBcAg蛋白能自主装配成大小不同的2种颗粒(即核心颗粒),大颗粒直径约34nm左右,小颗粒直径约30nm左右。同时,我们还观察到,该核心蛋白颗粒在体外可发生集聚现象。     

乙酰氨基半乳糖转移酶14(GALNT14)在毕赤酵母中的表达纯化及活性鉴定

将GALNT14全长编码区克隆到分泌型酵母表达载体pPIC9K中,构建重组载体pPIC9K-T14,经电转至毕赤酵母GS115中表达,使用G418筛选高表达重组菌,并对诱导条件进行优化,表达产物使用SDS-PAGE分析、Western blot鉴定、Sephadex G-100纯化、最后经HPLC检测活性。结果显示,在提供更好的供氧量条件下,用0.75％甲醇诱导可提高目的蛋白的表达量而降低杂蛋白的表达。培养上清液经SDS-PAGE检测显示目的蛋白分子量约64 kDa;Western blot结果显示在相应分子量处有一条特异性条带;利用Sephadex G-100成功分离纯化了目的蛋白;活性测定结果显示所获得的目的蛋白具有催化活性,为深入研究GALNT14的结构与功能奠定了基础。     

人源性乙酰胆碱受体α亚基胞外肽段的原核表达与血清学诊断研究

目的:利用原核系统获得重组乙酰胆碱受体(AchR)α亚基胞外肽段,通过血清学验证重组蛋白进行体外诊断可行性。方法:合成AchRα亚基胞外段全基因组核酸序列,构建pET-NusA-AchR-α融合表达载体,在原核系统内进行重组表达,以Western印迹和ELISA检测重组蛋白的抗原性和实用性。结果:原核重组Nus A-AchR-α蛋白具有强的免疫原性;ELISA检测200份临床阳性血清和100份健康人血清,阳性检出率达60%,阴性检出率为5%。结论:融合蛋白NusA-AchR-α可在原核系统内高表达,初步验证重组融合蛋白具有良好的抗原性和特异性。     

乙型肝炎病毒包膜M蛋白在巴斯德毕赤酵母中的表达

转化了乙肝病毒 preS2 S基因的重组巴斯德毕赤酵母菌株经甘油培养基充分增殖 ,然后转移到甲醇培养基中进行诱导表达。破碎细胞并提取胞内蛋白 ,经ELISA和WesternBlot检测 ,证明有 4株GS1 1 5 /HIS MUT 表达了HBVM蛋白。     

脑膜炎球菌P64K蛋白作为新型抗原载体的研究进展

P64K是来自奈瑟氏属脑膜炎球菌的一种外层膜蛋白,发现于脑膜炎恢复期患者和接种过古巴VAMENGOCBC疫苗的个体血清内。编码P64K蛋白的基因称lpdA,其编码序列长1782bp。P64K蛋白含593个氨基酸,由两个结构域组成,有二氢硫辛酸酰胺酰基脱氢酶活性。P64K是抗脑膜炎疫苗的候选者。因为P64K的高分子量和强免疫原性,被认为是一种很有前途的抗原载体 。     

CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定

目的：在杆状病毒昆虫细胞表达系统（baculovirus expression vector system,BEVS）中表达结核分枝杆菌蛋白CFP10-ESAT-6-MPB64,并鉴定其免疫原性。方法：目的基因CFP10-ESAT-6-MPB64连接到pFastBac转移载体并转化DH10Bac感受态,通过Tn7转座片段将目的基因转座到Bacmid中,得到Bacmid-CFP10-ESAT-6-MPB64穿梭载体,脂质体包被后转染Spodoptera frugiperda（Sf9）细胞收获病毒,病毒转染细胞后收集上清通过Co亲和层析纯化得到目的蛋白。纯化的蛋白免疫Balb/c小鼠并检测血清中特异性抗体滴度及PPD抗体,ELISA检测CFP10-ESAT-6-MPB64蛋白刺激脾脏细胞产生IFN-γ的浓度,MTT法检测目的蛋白对免疫后小鼠脾脏细胞的增殖作用。结果：CFP10-ESAT-6-MPB64在昆虫细胞中成功表达,纯化后蛋白纯度达90%以上,蛋白产量为42mg/L,纯化蛋白能有效刺激Balb/c小鼠产生抗体,提高小鼠脾脏细胞培养基中IFN-γ的含量,目的蛋白在1~50μg/ml之间对脾脏细胞有明显的促增殖作用。     

乙醇酸氧化酶基因在巴斯德毕赤氏酵母中的表达

将菠菜乙醇酸氧化酶基因片段克隆至表达载体pPIC3．5k。提取重组质粒,进行限制性酶切鉴定。重组质粒用Sal I酶切线性化,电导入法转化毕赤酵母(Pichia pastoris),在缺乏组氨酸的RDB平板筛选重组子,提取酵母的染色体基因组进行PCR扩增鉴定整合情况,用甲醇诱导表达。结果表明,SDS-PAGE电泳显示表达蛋白的分子量约为39．8kD,与文献报道的乙醇酸氧化酶分子量接近。酶的活力达到了40．8IU／g湿菌体,比不含有目的片断的对照菌酶活提高了17倍,确认了导入的乙醇酸氧化酶基因片段在酵母中高效表达。     

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

TOL plasmid in Pseudomonas aeruginosa PAO: thermosensitivity of self-maintenance and inhibition of host cell growth.

The TOL plasmid originally isolated in Pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of Pseudomonas, i.e., P. putida, P. fluorescens, and P. aeruginosa, except for a strain of P. aeruginosa, strain PAO. The same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. In addition, the transmissibility of the TOL plasmid from strain PAO to P. putida was low when the plasmid was modified by the donor. By using P. aeruginosa PAO carrying the TOL plasmid, the stability and genetic expression of the plasmid as well as its effect on the host cell growth were examined. Thus the self-maintenance of the plasmid was found to be thermosensitive. Furthermore, the TOL plasmid inhibited the growth of strain PAO at high temperature, accompanied by the formation of some filamentous cells. These thermosensitive properties of the TOL plasmid were host dependent and not exhibited in another strain of P. aeruginosa.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

High stability binding of poly(ADPribose) polymerase-like thermozyme from S. solfataricus with circular DNA

The poly(ADPribose) polymerase-like thermozyme from the hyperthermophilic archaeon S. solfataricus was found to bind DNA with high affinity and non-specifically. Binding was independent of base composition and length of the nucleic acid, and the protein showed a slight preference for the circular structure. By using pCMV-Neo-Bam plasmid as experimental model, the behaviour of the thermozyme upon binding with either circular or linear plasmid was analyzed. pCMV-Neo-Bam has a single HindIII site that allows to obtain the linear structure after digestion with the restriction enzyme. Intrinsic tryptophan-dependent fluorescence of poly(ADPribose) polymerase-like thermozyme noticeably changed upon addition of either circular or linear plasmid, showing the same binding affinity (K=2 x 10(9) M-1). However, experiments of protection against temperature and DNase I gave evidence that the thermozyme formed more stable complexes with the circular structure than with the linear pCMV-Neo-Bam. Increasing temperature at various DNA/protein ratios had a double effect to reduce the amount of circular DNA undergoing denaturation and to split the melting point towards higher temperatures. Nil or irrelevant effect was observed with the linear form. Similarly, DNase acted preferentially on the linear plasmid/protein complexes, producing an extensive digestion even at high protein/DNA ratios, whereas the circular plasmid was protected by the thermozyme in a dose-dependent manner. The complexes formed by archaeal poly(ADPribose) polymerase (PARPss) with the circular plasmid were visualized by bandshift experiments both with ethidium bromide staining and by labelling the circular plasmid with 32P. The stability of complexes was tested as a function of enzyme concentration and in the presence of a cold competitor and of 0.1% SDS. From the performed experiments, a number of 3-10 base pairs bound per molecule of enzyme was calculated, indicating a high frequency of binding. The presence of circular DNA was also able to increase by 80% the poly(ADPribose)polymerase-like activity, as compared to 25% activation induced by the linear pCMV-Neo-Bam.     

Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations

We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.