丝心蛋白基因分子克隆与表达的初步探讨

通过聚合酶链反应扩增丝心蛋白Ｃ亚基结构的基因，并将基克隆到融合蛋白表达载体ｐＲＩＴ２Ｔ质粒中得到ｐＲＩＴ２Ｔ－ＦＬ质粒，在大肠杆菌株Ｐ２３９２内进行表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和免疫印迹反应证明融合蛋白在大肠杆菌中得到了表达。     

人野生型MBL基因在大肠杆菌中的表达

为获得人MBL蛋白，并对其功能进行初步研究，用DNA重组法构建了组氨到标签融合原核表达质粒pET28(b)-MBL。将重组质粒转入大肠杆菌BL21（DE3），经IPTG在37℃条件下诱导培养，利用SDS－PAGE，Westem-blot检测目的蛋白的表达，用IMAC金属螯合层析柱对其进行纯化。成功地表达了重组MBL蛋白，纯化的MBL浓度约为844μg/mL，为制备MBL的基因工程抗体奠定了基础。     

[Bl0Glu，B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

采用PCR方法，将人胰岛素分子B链B10位His突变为Glu，在B24和B25位之间插入Asp．构建了[B10Glu，B24-Asp-B25]胰岛素原基因、利用通用型质粒pBV220构建表达载体，在大肠杆菌DH5a中表达．表达蛋白为包含体形式．约占菌体总蛋白的20％～30％．经过复性和凝胶过滤得到胰岛素原融合蛋白、用胰蛋白酶和羧肽酶B酶切．经DEAE离子交换和RP-HPLC纯化得胰岛素突变体类似物．用凝胶过滤法测定了蛋白质分子自身的缔合性质．用圆二色谱测定了构象变化、放射性免疫活性及受体结合活性测定结果表明，突变体分子缔合性明显下降．放免活性和受体结合活性分别约为人胰岛素的73．6％和146％。     

单克隆抗体-白介素2融合蛋白一级结构的确证

建立单克隆抗体-白介素2融合蛋白一级结构确证的方法.用基质辅助激光解吸飞行时间质谱测定单克隆抗体-白介素2融合蛋白的精确相对分子质量,毛细管等电聚焦方法测定其等电点,通过N-末端氨基酸序列的测定以及肽图分析,证实了抗体-白介素2融合蛋白一级结构表达的正确性,同时为单克隆抗体-白介素2融合蛋白的质量标准研究奠定了基础.     

融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响

将灵芝免疫调节蛋白(LZ-8)与免疫球蛋白G的Fc片段(IgG Fc)进行融合表达,通过Protein A亲和层析结合Superdex 75分子筛层析获得了高纯度LZ8-Fc.凝血活性实验结果表明,LZ-8与Fc融合表达在一定程度上影响了其免疫活性;利用分子动力学和圆二色光谱等技术研究了LZ8-Fc的蛋白折叠及组装方式,发现LZ8-Fc的组装方式与LZ-8相同,但是N末端的LZ-8可能影响下游IgG Fc片段的正确折叠,表明在融合蛋白制备过程中,位于N末端的蛋白对下游蛋白的正确折叠起到重要作用,这为融合蛋白制备技术的优化及应用过程中的稳定提供了重要的理论依据.     

具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化

用PCR法从质粒pHB3中扩增了人红细胞带3蛋白胞质片段(CDB3)基因.PCR产物经限制性内切酶切割后与多克隆位点处带有编码6个组氨酸序列的高效表达载体pET28b连接,构建为重组子pCDBHistag.重组子经酶切及序列测定后在大肠杆菌BL21(DE3)中获得高效表达,可溶性目的蛋白占菌体总蛋白的40%左右.C端带有6个连续组氨酸的带3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度,而且简化了目的蛋白的纯化过程.经一步螯合Ni²⁺的亲和层析获得了电泳纯的带3蛋白胞质片段融合蛋白.活性测定结果表明,带3蛋白胞质片段融合蛋白能够抑制醛缩酶(Aldolase)活性的70%,与文献报道的人红细胞内带3蛋白胞质片段具有相同的功能.     

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.     

志贺氏毒素B亚单位在大肠杆菌细胞表面表达的研究

本文通过寡核苷酸引导的定向诱变,将志贺氏毒素B亚单位的三个部分(Stx17B,Stx27B和StxB)融合至暴露在细菌表面的LamB蛋白的第153和154氨基酸之间的BamHⅠ位点。免疫印迹指出,B亚单位的三个部分作为与LamB的融合蛋白能在大肠杆菌中表达。间接免疫荧光和免疫电子显微镜分析表明:融合蛋白LamB/Stx17B和LamB/Stx27B能表达在大肠杆菌细胞表面,而融合蛋白LamB/StxB则不能在大肠杆菌表面表达,它聚集在细胞间质部分,对宿主细胞有毒性。将保护性抗原表达于细菌表面,这为构建预防志贺氏痢疾杆菌的菌苗后选株提供了一个新的途径。     

肠道病毒71型外壳蛋白VP1在大肠杆菌中的表达

将扩增得到的肠道病毒71型外壳蛋白VP1基因克隆到测序载体pGEM-T,测序验证该序列为目的片段后,将目的基因克隆到原核表达载体pGEX-5x-1中,转化大肠杆菌BL21,IPTG诱导表达,产物经SDS-PAGE分析和Western blot验证。结果表明,在经IPTG诱导的BL21中检测到分子量与预期大小相符的大约60 kDa的融合蛋白。利用表达产物作为抗原,对EV71感染病人阳性血清的检测初步证实,重组蛋白VP1可以作为检测EV71感染的检测用抗原。     

猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定

以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,westem blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.     

粘着斑激酶的原核表达、纯化及抗体的制备

粘着斑激酶(focal adhesion kinase,FAK)是细胞质内单亚基非受体型酪氨酸激酶,通过各种信号途径参与调节细胞生长、发育、黏附、细胞骨架重组、转化、扩散和迁移等过程.采用PCR方法,从Flag-FAK质粒中克隆编码FAK C端273个氨基酸的基因片段,构建FAK融合蛋白原核表达载体pET28a( )/FAK,进行原核表达与蛋白纯化,取纯化的FAK蛋白免疫小鼠,制备FAK抗血清.结果表明构建的表达FAK C端功能结构域的原核表达质粒pET28a( )/FAK,经过BL21(DE3)大肠杆菌表达、镍亲和层析柱纯化,获得相对分子质量约33 kDa的融合蛋白,并利用小鼠制备了多克隆抗体,EL ISA检测显示该抗体有较高效价.荧光免疫结果显示此多克隆抗体与FAK蛋白特异性结合,为进一步研究神经细胞中FAK的作用机制奠定了基础.     

SPAP2基因的克隆、融合表达及分离纯化

本文构建的表达载体pGex-2T-SPAP2CT在大肠杆菌中表达出可融性蛋白, 其分子量为46 000, 经纯化后得到产率为10%、 纯度大于90%的GST-SPAP2CT蛋白.     

Properties of GST-CALM expressed in E. coli

Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.     

Phage Displayed HBV Core Antigen with Immunogenic Activity

Hepatitis B is a major public health problem worldwide, which may lead to chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The hepatitis B core antigen (HBcAg) is one of the major viral proteins, which forms the inner core of hepatitis B virus (HBV) particles. In this study, filamentous bacteriophage M13 was genetically modified to display the polypeptides of HBcAg in order to develop an alternative carrier system. HBcAg gene was inserted into the minor coat protein (pIII) gene of M13, and HBcAg was expressed on the phage surface as a whole protein. Antigenicity and immunogenicity of HBcAg were tested by immunizing BALB/c mice three times with HBcAg-displaying recombinant phages. After successful immunization, one of the mice with high antibody titer to HBcAg was selected for fusion, and four monoclonal antibodies specific for HBcAg were developed. This result showed that HBcAg-displaying recombinant bacteriophages are immunogenic and can potentially be used for the development of monoclonal antibodies.     

Expression of CTB-human Insulin(BA) Fusion Protein in Gynostemma Pentapyhllum Makino Callus Cells and Its Hypoglycemic Effect in Mice

The plant expression vector of choleratoxin B subunit(CTB)-human insulin(BA) fusion protein pBI121/(CTB-BA) was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express CTB-human insulin fusion protein was constructed and its hypoglycemic effect was evaluated in mice. The plant expression vector pBI121/(CTB-BA) was digested with both BamI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/(CTB-BA) via the freeze tha-wing method, then CTB-BA gene was integrated to G Pentapyhllum Makino callus cells by co-culturing the cells with the transformed LBA4404 strain. The transformed G Pentapyhllum Makino callus cells were identified by DNA se-quence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice.The sequences of synthetic CTB and human insulin genes(BA) were completely identical to those designed. Restriction map proved that the length of gene fragment in-serted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed. The result of RT-PCR was consistent with that expected. The ex-pressed product showed a specific band with a relative molecular mass of 17000 by Western-blot. The human insulin expression amount was 6.03 μIU/mL according to the ELISA result.The animal test showed that only the G Penta-pyhllum Makino callus cell line itself showed activity in decreasing the blood sugar of mice, however, the activity of the transformed G Pentapyhllum Makino callus cells was much higher. The plant expression vector pBI121/(CTB-BA) was constructed and expressed in the G Pentapyhllum Makino callus cells successfully for the first time. The trans-formed G Pentapyhllum Makino callus cells showed high activity in decreasing the blood sugar of mice. This study developed a new way for the development of oral administration insulin.     

Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E.coil

The entire gene of carboxyltransferase(CT) domain of acetyl-CoA carboxylase(ACCase) from Chinese Spring wheat(CSW) plastid was cloned firstly,and the 2.3 kb gene was inserted into PET28a+ vector and expressed in E.coil in a soluble state.The (His)6 fusion protein was identified by SDS-PAGE and Western blot.The recombinant protein was purified by affinity chromatography,and the calculated molecular mass(Mr) was 88000.The results of the sequence analysis indicate that the cloned gene(GeneBank accession No.EU124675) was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid.The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides,and further to screen new herbicides.     

Expression of a restricted fragment of staphylococcal tetracycline resistance determinant as a fused product in Escherichia coli

Tetracycline resistance (TcR) plasmid pNS1, a deletion derivative constructed from staphylococcal plasmid pTP5, carries a tet determinant which specifies a TcR protein (TET) with a molecular weight of 50 kilodaltons (kDa). In order to express the pNS1-encoded TET as a fused product, a 0.8 kilobase pairs fragment containing 57.1% of tet determinant was inserted into a chloramphenicol resistance determinant. From the nucleotide sequence, it is deduced that the fusion protein (designated CAT'-TET') is a 53 kDa protein composed of 472 amino acids in which the 199 and 262 amino acids are derived from CAT and TET, respectively. Although the molecular weight of CAT'-TET' obtained from the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (42 kDa) was not in agreement with its predicted weight (53 kDa), the ratio of TET' segment to the fusion protein (22 kDa/42 kDa) corresponded almost exactly to that deduced from the nucleotide sequence (29 kDa/53 kDa). The expression of CAT'-TET' in Escherichia coli caused a rapid decrease in growth rate and in the number of viable cells. This result is thought to be due to the toxic effect of CAT'-TET' on the cell membrane.     

Molecular and Computational Approaches to Characterize Thermostable Laccase Gene from Two Xerophytic Plant Species

Laccases are blue multicopper oxidases that carry out single electron transfers in the oxidation of phenols to quinones. In plants, they confer structural stability to the cell wall. Thermostable laccases were identified in xerophytes Cereus pterogonus and Opuntia vulgaris that could be used in biotechnology and industrial processes. Polyclonal anti-laccase antibodies were generated against purified laccase enzyme isoforms capable of 98–99 % inhibition of the catalytic activity. Antibodies raised against lower molecular weight isoforms inhibited 70 % of the catalytic activity of higher molecular forms. Only 20 % inhibition was noted when assayed in reverse. A partial gene sequence of thermostable xerophytic laccase comprising 712 and 880 bp was identified employing cDNA as template. The nucleotide sequence was submitted to GenBank. The gene sequence was in silico translated into protein sequence and a 3-D structure was predicted using I-Tasser and Genesilico online servers that justified the experimental observations. Anti-laccase antibodies and nucleotide gene sequence of this thermostable plant laccase can be utilized for predicting laccase antigenic sequences and for cloning and expression of the thermostable eukaryotic laccase.