Prokaryotic expression and identification of a small peptide of osteopontin

AIM: To express osteopontin 13 peptide (OPN 13) in E.coli, and to test the biological activity of the purified products. METHODS: cDNA fragments containing RGD sequences were cloned into prokaryotic expression vector pET-32c(+) including His coding sequence to construct pET-32c-OPN 13 plasmid. E.coli DH5α transformed by pET-32c-OPN 13 plasmid was induced by IPTG at different concentrations for different times to identify the optimal induction condition. Expressed His-OPN 13 fusion protein was purified via Ni-NTA His Bind Resin metal chelation chromatography, and detected by VSMCs adhesion and migration analysis. RESULTS: His-OPN 13 fusion protein was expressed in soluble manner. The fusion proteins were purified via Ni-NTA His Bind Resin affinity chromatography. His-OPN 13 fusion protein specifically inhibited adhesion and migration of VSMCs stimulated by osteopontin in dose-dependent manner. CONCLUSION: The OPN 13 peptide is successfully expressed in E.coli DH5α. The purified His-OPN 13 fusion protein could inhibit the adhesion and migration of VSMCs stimulated by osteopontin.     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

罗汉果葡萄糖基转移酶基因的克隆及原核表达

 从罗汉果（Siraitia grosvenorii）转录组中获得一条与罗汉果甜苷Ⅴ生物合成相关的葡萄糖基转移酶（UDPG）的unigene片段,以罗汉果授粉后70 d的果实RNA为模板,利用RACE和RT-PCR技术克隆UDPG全长基因,将克隆得到的SgUDPG1基因连接到原核表达载体pEASY-E1上,构建融合表达载体,转化到大肠杆菌BL21（DE3）,通过IPTG诱导表达,重组蛋白纯化,SDS-PAGE检测表达产物以及Western-blotting和质谱鉴定蛋白产物。结果表明,获得了1条SgUDPG1,全长为1 959 bp,开放阅读框ORF为1 365 bp,编码1条454 aa的肽链,理论分子量为51.2 kD,等电点为5.39,具有植物中次生代谢产物糖基转移酶特有的保守结构域PSPG-box motif。SgUDPG1在授粉后50 d和70 d的果实中表达逐渐升高,是对照授粉后3 d的5.16倍和13.12倍,与果实中甜苷Ⅴ含量呈相同趋势。此基因的ORF可以在大肠杆菌中表达,并且可以纯化出比理论分子量大5.3 kD的融合蛋白,通过Western-blotting和质谱鉴定,确定该蛋白属于罗汉果葡萄糖基转移酶。     

Synthesization,expression, purification and activity assay of hIGF-1

AIM: To express the synthesized human insulin like growth factor I （hIGF-1） gene in E.coli with high expression level and explore the way to increase the efficiency of factor Xa cleavage. METHODS: The gene of hIGF-1 was designed and synthesized according to the preference of E.coli. A fusion protein with a recognized site of factor Xa between CBD (cellulose binding domain) and hIGF-1 was expressed and purified by cellulose affinity chromatography. MTT method was used to assay the bioactivity of CBD-IGF fusion protein. hIGF-1 was released by factor Xa. In order to improve the sensitivity of fusion protein to factor Xa, the short flexible peptide （Gly-Thr-Gly- Gly-Gly-Ser-Gly） was added before the recognized site of factor Xa. RESULTS: SDS-PAGE results indicated that the CBD-IGF fusion protein was expressed and purified . Biological assay results indicated CBD-IGF fusion protein could promote the growth of NIH3T3 cell. The short flexible peptide （Gly-Thr-Gly-Gly-Gly-Ser-Gly）, which was added before the recognized site of factor Xa, improved the sensitivity of fusion protein to factor Xa. CONCLUSION: CBD-IGF fusion protein with bioactivite are expressed and purified. The amio acid sequences changes between the site recognize of factor Xa can help to improve the cleavage efficiency of Factor Xa.     

Cloning and expression of the metalloproteinase domain of human von Willebrand factor-cleaving protease

AIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6×His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function.     

Construction of lymphocyte subtracted cDNA library of unstable angina

AIM:To construct a subtracted cDNA library of differentially expressed genes in human unstable angina lymphocytes.METHODS:Suppression subtractive hybridizations (SSH) were performed between the patients with unstable angina pectoris and stable angina pectoris. Lymphocyte RNA, the obtained forward and reverse cDNA fragments were directly inserted into T/A cloning vector and transformed into E.coli JM 109 to construct a subtractive cDNA library. The inserting fragments were screened by blue and while blot screening and bacterium liqulid PCR.RESULTS:Each subtractive cDNA library contained more than 2000 positive bacteria clones. Most of them distributed between 200-600 bp inserts.CONCLUSION:The library is efficient and lays solid foundation for screening and cloning new and specific expressed genes in unstable angina lymphocyte RNA.     

欢迎订阅2006年下列期刊

AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10. V     

Cloning and expression of mouse canstatin cDNA in E.coli

AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.     

Construction of prokaryotic expression vector of human angiogenesis inhibitor arresten and its expression in E.coli

AIM:To construct prokaryotic expression vector of human angiogenesis inhibitor arresten gene and express recombinant arresten in Escherichia coli.METHODS:Human arresten gene was amplified from recombinant plasmid pGEM-Arr with polymerase chain reaction (PCR), and then cloned into prokaryotic expression vector pRSET by means of recombinant gene technology. The recombinant plasmid pRSET-Arr was transformed into E.coli BL21(DE3), and recombinant arresten was expressed in the bacteria under induction of IPTG. The expressed products were detected by SDS-PAGE analysis.RESULTS:Restriction analysis indicated that the arresten gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant arresten was successfully expressed in Escherichia coli; its molecular weight was about 26 kD and its amount was approximately 30% of total bacterial proteins.CONCLUSION:The successful construction of prokaryotic expression vector containing human arresten gene and the effective expression of recombinant arresten in Escherichia coli laid the foundation for further study on its biological functions.     

韧皮部特异启动子驱动密码子优化的抗菌肽D基因的植物表达载体构建

根据177个GenBank中登录的柑橘编码蛋白密码子用法的分析结果,优化并重新设计和合成了含柑橘偏爱密码子、对柑橘黄龙病有杀灭作用的柞蚕抗菌肽D基因(命名为CAPD),克隆入pUC19克隆载体并经测序验证后,获得了含新抗病基因的重组质粒pUC19-CAPD。用限制性内切酶BamHI和SacI双酶切pUC19-CAPD克隆载体和pBI121植物表达载体的质粒DNA,回收pUC19-CAPD克隆载体中的CAPD基因小片段和pBI121植物表达载体中去掉GUS报告基因的大片段,经连接、转化和鉴定后,构建了由CaMV35S组成型启动子(35SP)驱动CAPD目的基因的新植物表达载体(命名为pHZ05);用限制性内切酶BamHI和HindIII双酶切含笋瓜韧皮部特异启动子(PSP)的pUCm-PSP克隆载体和pHZ05植物表达载体的质粒DNA,分别回收pUCm-PSP克隆载体中的PSP小片段和pHZ05植物表达载体中去掉CAPD目的基因上游35SP的大片段,经连接、转化和鉴定后,构建了由PSP驱动CAPD目的基因的新植物表达载体(命名为pHZ06)。利用细胞感受态法直接将2个由不同启动子驱动的含CAPD目的基因的新重组植物表达载体分别导入根癌农杆菌LBA4404、GV3101、EHA105和发根农杆菌Ri15834等4个农杆菌菌株中,为利用农杆菌介导的遗传转化技术培育抵抗由韧皮部传导的毁灭性和检疫性病害柑橘黄龙病的新种质奠定了基础。     

山东寿光地区番茄黄化曲叶病毒外壳蛋白基因克隆及其在大肠杆菌中的表达

利用双生病毒简并引物PA/PB,对山东寿光地区保护地疑似感染番茄黄化曲叶病毒（TYLCV）的番茄植株进行PCR检测,结果证明番茄病叶由TYLCV侵染所致。以提取的病叶总DNA为模板,扩增得到长约770 bp的TYLCV-CP基因片段,克隆至pEASY-T1 Simple载体,然后用Xho I和EcoR I将其切下并插入到pET-32a表达载体中,构建了寿光地区TYLCV分离物的外壳蛋白基因原核表达载体,转入大肠杆菌BL21（DE3）,经IPTG（异丙基-β-D-硫代吡喃半乳糖苷）诱导获得了50 kD重组蛋白,Ni2+-NTA亲和层析纯化和Western印迹分析证明目的蛋白为TYLCV外壳蛋白,且具有良好的抗原活性。     

黄瓜动蛋白特异肽段的原核表达和多克隆抗体制备

 以黄瓜果实总RNA为模板,通过RT-PCR获得了动蛋白基因CsKF1和CsKF2的cDNA序列,进行测序,构建T-easy载体。SMART在线预测CsKF1和CsKF2均含有动蛋白（Kinesin）约340个氨基酸残基的马达区保守序列（moter domain）。体外表达蛋白的马达区和非马达区,将带有His标签的CsKF1-N、CsKF2-C、CsKF1-M、CsKF2-M融合蛋白通过E. coli BL21（DE3）表达,摸索不同诱导条件下目的蛋白的表达和纯化。其中His-CsKF1-N、His-CsKF2-C、His-CsKF1-M原核表达蛋白的分子量分别为25、30和40 kD,均以0.1 mmol · L^-1 IPTG,22 ℃ 6 h诱导表达效果最好。His-CsKF2-M原核表达蛋白的分子量约38 kD,以18 ℃ 8 h诱导表达效果最好。融合蛋白His-CsKF1-N和His-CsKF2-C经过亲和纯化后作为抗原,通过5轮免疫注射兔子后,取心脏血作为抗血清,通过AminoLink Plus kit试剂盒亲和纯化得到多克隆抗体anti-CsKF1-N和anti-CsKF2-C。经过Western blot分析表明多克隆抗体具有特异性,可与抗原特异结合。     

中矮1号梨砧木(S_2)PGIP基因的克隆及序列分析

利用RT-PCR技术,根据GenBank中梨属PGIP基因序列设计1对特异引物,以梨矮化砧木S2叶片总RNA为模板,克隆到1条约1100bp的cDNA片段,将其与pMD18-Tvector连接后转化Escherichia coli JM109,对筛选到的阳性克隆进行序列测定并使用生物信息学方法对所得结果进行综合分析。结果表明,克隆片段为梨PGIP基因,该cDNA编码330个氨基酸,预测分子量为36388ku,第1～24个氨基酸残基是信号肽。与梨属其它种中已获得的PGIP核苷酸序列开放阅读框(Open reading frame,ORF)的同源性为97.5%～99.6%,氨基酸序列的同源性为97.6%～99.1%。     

‘三棱榄''橄榄果实香气成分分析

1 材料与方法选取广东优良鲜食橄榄品种‘三棱榄’,2001年12月6日采样,采用固相微萃取法(SPME)富集香气成分(鲜橄榄果肉于15℃下捣碎后取样1.0 g放入4 mL聚四氟乙烯硅橡胶垫密封螺口玻璃瓶中,插入100μm聚二甲基硅氧烷纤维头于室温25-30℃顶空取样2 h),用美国Finnigan TRACE GC-MS气相色谱-质谱联用仪进行分析。气相色谱柱为DB-1弹性毛细管柱30 m×0.25 mm,载气为He(99.99％),流速1.0mL/min。程序升温从40℃开始先保持10 min,后以2℃/min的升温速率升至150℃保持10 min。质谱条件:电子能量70 eV,离子源温度250℃,质量范围35-450 aum,不分流进样。2002年12月18日采样重复分析。     

葡萄A病毒四川分离物的外壳蛋白基因克隆与原核表达

葡萄A病毒（Grapevine Virus A,GVA）是葡萄病毒属（Vitivirus）的典型种,在世界葡萄产区广泛分布。采集10株“藤稔”葡萄成熟枝条,使用6种葡萄病毒ELISA试剂盒检测发现10个样本中有6个感染4种不同的葡萄病毒。以GVA的ELISA阳性植株为材料进行RT-PCR扩增,首次获得了GVA四川分离物SL10的完整外壳蛋白基因（CP）。该基因全长597 bp,将其与GeneBank收录的15个GVA分离物的CP序列进行比对和构建系统进化树。把不同地理起源的GVA分离物分成2个变异组;其中Ⅰ组包括3个分离物(与Ⅱ组的其他分离物只有75.9%～80.1%的序列同一性);其余的13个分离物组成Ⅱ组（组内分离物具有84.4%～99.5%的序列同一性）。构建了GVA CP的原核表达质粒PET-30-GVAcp并转化BL21菌株,经IPTG诱导,目的基因得到了大量表达。     

Cloning and expression of human plasminogen kringle 5 gene in E.Coli

AIM: Constructing plasmid that expresses human plasminogen kringle 5 gene to analyze the gene expression in E.coli. METHODS: The gene of human plasminogen kringle 5 was inserted into plasmid PBV220 EcoRI site by gene manipulation techniques and was transformed to E.coli TGI. The gene expression was observed by SDS-PAGE. RESULTS: Expression vector PBVK5 was constructed, and human plasmingen kringle 5 gene product was obtained at 42℃ induction. CONCLUSION: Expression product of human plasminogen kringle 5 gene was soluble form of proteins, and the expression amount was 9.8% in E.coli TGI total proteins.     

Prokaryotic expression of Staphylococcus aureus Panton-Valentine leukocidin and its cytolytic activities to human polymorphonuclear neutrophils

AIM:Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus epidemiologically associated with the often-lethal necrotizing pneumonia. Until now, the mechanisms of pathogenesis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult. In the present study, we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro, which provides an experimental basis for the further studies of its biological function and its toxicity in pneumonia. METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by high-fidelity PCR was cloned into prokaryotic expression vector pET22b(+), and the vector was transformed into BL21 (DE3)plysS to construct a prokaryotic expression system. The integrity of the opening-reading frame of each construct was verified by DNA sequencing. The recombinant PVL (rPVL) was induced by1.0 mmol/L IPTG. The expressed products were identified by SDS-PAGE and the fusion proteins (6His-LukS-PV and 6His-LukF-PV) were purified from lysates of transfected E. coli cells by affinity chromatography on nitrilotriacetic acid columns. The cytolytic activity was tested by incubation of rPVL with human polymorphonuclear neutrophils (PMNs) in vitro. RESULTS:The nucleotide sequence of the cloned PVL gene was the same as that of reported in GenBank. E. coli BL21 (DE3)plysS containing recombinant vectors grow at 37℃ causes some proteins to accumulate as inclusion bodies, while incubation at 30 ℃ led to a significant amount of soluble active proteins which accounted for about 31.7% of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD, and the LukF-PV protein was about 35 kD. The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated. CONCLUSION:The genes of lukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+) and expressed in E. coli respectively, which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.     

Cloning,expression and characterization of sLAG-3-Ig

AIM: To obtain a vaccine with sLAG-3 as immunoadjuvant and investigate its biologic activity in order to establish the safe and effective way for asthma as one of the specific immunotherapy. METHODS: The coding sequence of LAG-3 was amplified by polymerase chain reaction, the expression vector pcDNA-sLAG-3-Ig was constructed by inserting the PCR products of sLAG-3 and Fc sequence of IgG. With electroporation transfection, pcDNA-sLAG-3-Ig was transfected into COS-7 cells and its biologic activity was investigated by Western blotting analysis. RESULTS: By temperature induction, the LAG-3-Ig was highly expressed in E.coli DH5α. LAG-3-Ig fusion protein was observed by SDS-PAGE and Western blotting, the results showed that the LAG-3-Ig protein was an antagonist of the IL-4-induced synthesis of IgE in B cells. CONCLUSION: A new vaccine with sLAG-3 as immunoadjuvant was obtained. It could inhibit synthesis of IgE in B cells. Thus, LAG-3-Ig would be hopeful to establish the safe and effective way for asthma as one of the specific immunotherapy.     

桦褐孔菌纤维素酶基因的克隆与原核表达

成功获得了桦褐孔菌(Inonotus obliquus)JL 01菌株的内切葡聚糖酶(IO-EG)和β-葡萄糖苷酶(IO-BGL)的cDNA全长序列.eg2基因cDNA序列全长(ORF)为1149 bp,编码382个氨基酸,bgl2基因cDNA序列全长(ORF)为2583 bp,编码860个氨基酸;成功构建了30a-eg2和30a-bgl2大肠杆菌表达菌株,诱导培养后,SDS-PAGE电泳分析表明两个菌株均表达蛋白,且蛋白以包涵体形式存在,为桦褐孔菌高效外源基因表达系统的建立提供了基础.