新克隆的硝基还原酶基因NOR1的表达及其产物的纯化

背景与目的：NOR1是与鼻咽癌密切相关的抑瘤／易感基因候选者之一,本实验旨在构建NOR1基因原核表达载体,在大肠杆菌中表达并纯化NOR1基因所编码的蛋白。方法：抽提人鼻咽正常组织中总RNA,利用RT－PCR扩增NOR1基因全长,经BamH I、Xho I双酶切后插入同样经BamH I、Xho I双酶切后的pGEX-4T-2质粒,构建表达载体pGEX-4T-2/NOR1重组表达质粒;转化大肠杆菌Jm105,用异丙基－β-D硫代半乳糖苷（isopropyl-thiogalactoside,IPTG）诱导pGEX-4T-2/NOR1/Jm105表达GST融合蛋白后,采用Western blot验证,并用GST琼脂糖亲和层析柱对目的蛋白进行纯化。结果：酶切鉴定和测序验证成功地构建了NOR1基因原核表达载体,经十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate polyacrylamide gel electrophoresis,SDS-PAGE）分析在大肠杆菌Jm105中成功诱导表达了一分子质量约为74kDa的融合蛋白;该融合蛋白存在于细菌裂解液的上清和沉淀中,Western blot证实表达获得了成功;并利用亲和层析法获得了纯化蛋白。结论：成功获得了高效表达NOR1基因的原核表达产物,通过对该融合蛋白的纯化为它的多抗制备奠定了基础。     

肿瘤-睾丸抗原LAGE-1基因的原核表达纯化和抗血清制备

目的:构建人肿瘤-睾丸抗原LAGE-1的原核表达质粒,表达、纯化LAGE-1融合蛋白并制备GST-LAGE-1多克隆抗体。方法:逆转录聚合酶链反应(RT-PCR)方法扩增人肝癌组织LAGE-1基因3′端片段,连入原核表达载体pGEX-4T-1( ),构建原核表达质粒pGEX-LAGE-1并测序。将该质粒转化大肠埃希菌BL21(DE3)进行诱导表达,经包涵体透析复性和谷胱甘肽巯基转移酶纯化系统分离纯化目的蛋白。用纯化的融合蛋白GST-LAGE-1免疫新西兰大白兔,间接ELISA法测定抗体效价,饱和硫酸铵沉淀法纯化抗体,并采用Western blot检测GST-LAGE-1多克隆抗体特异性。结果:RT-PCR扩增得到LAGE-1基因3′端264bp片段,测序结果与GenBank公布序列一致;成功构建pGEX-LAGE-1原核表达质粒,含有该质粒的大肠埃希菌经IPTG诱导后表达相对分子质量约为36×103的蛋白,经包涵体复性和GST融合蛋白纯化系统纯化,得到重组蛋白GST-LAGE-1,纯度达90%;制备兔抗GST-LAGE-1抗体,纯化的抗体经Western blot证实可与目的蛋白特异性结合。结论:获得了纯化的肿瘤-睾丸抗原LAGE-1重组蛋白及其特异性多克隆抗体,为进一步研究LAGE-1抗原在肿瘤免疫治疗中的作用奠定基础。     

人NY-ESO-1基因的原核表达、纯化和初步应用

目的：克隆人NY-ESO-1基因，进行原核表达和分离纯化，并初步应用于肝癌患者血清NY-ESO-1抗体的检测。方法：通过RT-PCR技术从人肝癌组织中扩增NY-ESO-1基因片段，插入原核表达载体pGEX4T-1( )中，构建重组表达质粒pGEx／ESO1，导入大肠杆菌BL21(DE3)，诱导目的蛋白表达，经包涵体透析复性和GSTrap亲和层析纯化目的蛋白，Westem blot鉴定。应用间接ELISA方法初步检测50例肝癌患者血清和20例正常人血清NY-ESO-1抗体的表达。结果：扩增得到NY-ESO-1基因3′端276bp片段，与GeneBank公布序列一致，构建的pGEX／ESO1原核表达质粒经IPTG诱导，在大肠杆菌中表达分子量约36kD的GST-ESO1融合蛋白，纯化后蛋白纯度为90％。50例肝细胞癌患者血清中4例检测到NY-ESO-1抗体(8％)，正常人血清均为阴性。结论：成功构建pGEX／ESO1重组表达质粒，得到有活性的NY-ESO-1融合蛋白，对肝癌患者血清NY-ESO-1抗体的检测有-定的应用价值。     

PEP-1-VP3融合蛋白的原核表达及纯化

目的:构建融合蛋白PEP-1-VP3的原核表达质粒,并进行诱导表达和蛋白纯化。方法:通过PCR方法自PGEX-6P-1/TAT-VP3质粒中扩增VP3基因,运用靶向克隆酶,将VP3基因插入到线性载体pET15b-PEP-1,构建重组表达质粒pET15b-PEP-1-VP3,经测序证实构建成功后转化E.coli Rosetta,表达PEP-1-VP3融合蛋白,经Ni-NTA树脂亲和层析,纯化产物进行SDS-PAGE及Western blot分析鉴定。结果:成功构建PEP-1-VP3融合蛋白原核表达载体,表达并纯化了分子量为17.6kD的PEP-1-VP3融合蛋白。结论:PEP-1-VP3融合蛋白原核表达质粒的构建及目的蛋白的表达、纯化,为体内应用PEP-1-VP3融合蛋白进行抗肿瘤研究提供了理论基础。     

人肺腺癌TLE1 N端Q结构域片段在原核系统表达纯化及其多克隆抗体的制备

背景与目的 TLEI是参与Wnt、Notch以及EGFR信号通路调控的一种重要蛋白.TLE1 N端Q结构域片段通过介导自身寡聚化及与LEFl结合发挥调控作用.本实验旨在构建人TLE1 N端Q区基因在大肠杆菌的融合表达载体,制备并纯化人TLEI N端Q结构域蛋白片段,制备TLEl Q结构域多克隆抗体.方法 以人肺腺癌cDNA库为模板聚合酶链反应(polymerase chain reaction, PCR)特异性扩增TLE1-Q(1-136)基因序列,与pGEX-4T-1质粒连接后转化感受态大肠杆菌E.coli.以异丙基-β-D-硫代半乳糖苷(isopropyl-β-D thiogalactoside,IPTG)诱导表达产生融合蛋白GST-TLEI-Q(1-136).经亲和层析,Thrombin酶切,FPLC纯化,SDS-PAGE鉴定目的蛋白TLE1-Q(1-136).免疫家兔,制备多克隆抗体.结果 测序证实重组表达质粒中的人TLE1 N端Q结构域基因序列正确,成功构建表达型重组质粒pGEX-4T1-TLE1-Q重组质粒转化大肠杆菌c+后,经诱导,重组蛋白GST-TLE1-021-136)得到表达.SDS-PAGE鉴定示纯化蛋白为目的蛋白人TLEl N端Q结构域片段TLE1-Q(1-136).免疫家兔后收获抗血清,ELISA显示抗体效价为1:20000,具有高度特异性.免疫印迹结果显示,制备的多抗可与TLEI.021.136)蛋白特异性结合.结论 成功构建了人TLE1 N端Q结构域重组融合蛋白表达质粒pGEX-4T1-TLE1-Q表达纯化了稳定可溶TLE1 N端Q结构域蛋白TLE1-Q(1-136),制备了人TLE1 N端Q结构域蛋白片段多克隆抗体,为进一步研究TLE1在肺癌生成中的作用奠定了基础.     

人B细胞成熟抗原的克隆、表达及纯化

目的构建人B细胞成熟抗原（BCMA）原核表达质粒,表达BCMA融合蛋白。方法通过RT—PCR方法从人B淋巴瘤Raji总RNA中扩增BCMA的全长cDNA,并插入原核表达载体PGEX4T-1的BamHI和NotⅠ酶切位点,构建原核表达载体PGEX4T-1-BCMA,利用酶切和序列分析方法验证所构建质粒的正确性。在大肠杆菌中表达BCMA融合蛋白,并经谷胱甘肽-S-转移酶（GST）亲和柱纯化。纯化后的产物经过SDS—PAGE、Westernblot检测目的蛋白的表达情况。结果Bam HI／Notl双酶切证明重组质粒中含有目的大小的BCMA基因片段,序列分析证明重组质粒中插入片段的碱基序列均完全正确。将所构建的PGEX4T-1-BCMA质粒转染感受态DH5ct,IPTG诱导表达后超声裂解菌体,上清经GST亲和纯化,经SDS—PAGE、抗GST和BCMA抗体Westernblot分析,证实融合表达蛋白为GST—BCMA融合蛋白。结论成功构建了BCMA原核表达载体,重组质粒能够在原核表达系统中可溶性表达GST—BCMA融合蛋白,为进一步研究BCMA的生物学功能奠定了基础。     

肺腺癌耐药相关基因BC006151原核表达载体的构建及蛋白表达

背景与目的肺癌细胞对化疗药物的耐受是肺癌患者生存率低的重要原因之一。我们在前期研究中发现了一条肺腺癌耐药相关的基因BC006151,本研究拟构建肺腺癌耐药相关基因的原核表达载体PGEX-4T-1-BC006151,诱导其表达及鉴定目的蛋白,从而揭示该基因与肺腺癌耐药的关系。方法以RNA为模板RT-PCR扩增,产物与质粒PGEX-4T-1用BamHI和EcoRI双酶切,用T4DNA连接酶将二者连接,连接物转化DH5α菌,重组克隆菌经测序鉴定确认。将带有目的片段的重组克隆载体转化BL21表达菌,SDS-PAGE电泳检测表达产物,Western blot检测GST融合蛋白表达。结果经酶切鉴定及DNA测序,重组质粒中已插入了目的基因片段,与GenBank中公布的BC006151基因序列一致。重组克隆载体经BL21表达菌表达,获得40KD的条带(其中目的蛋白13KD,GST标签26KD)。结论成功构建了肺腺癌耐药相关基因BC006151原核表达载体,并成功诱导了目的蛋白表达,GST亲和纯化,为进一步制备该基因的多克隆、单克隆抗体奠定了基础。     

人热休克蛋白70的原核表达和纯化的实验研究

目的在大肠杆菌中表达人源性热休克蛋白70基因并纯化表达产物。方法以人HSP0 cDNA为模板,采用PCR方法进行扩增,将PCR产物克隆到表达载体pET30a上,将重组质粒pET30a-HSP70转化大肠杆菌BL21上,经IPTG诱导后,表达产物进行SDS-PAGE及Western blot验证并纯化。结果应用PCR方法扩增出约1900bp的目的片段;经酶切鉴定和DNA测序证实,HSP70基因成功地克隆到原核表达载体pET-30a上;转入重组质粒的大肠杆菌经IPTG诱导后,进行SDS-PAGE,发现在分子量约为70kD处有蛋白表达,Western blot证实其为目的蛋白,纯化后的HSP70纯度达到90%以上。结论在大肠杆菌中已成功地表达了HSP70蛋白并且经过纯化,为研究HSP70的结构、功能及临床应用提供了必要条件。     

hTERT原核重组质粒的表达

目的本课题拟观察原核重组质粒pGEX-4T-1-hTERT在BL21细菌中的表达，为进一步探讨以hTERT为靶点的肿瘤基因免疫治疗提供实验依据。方法IPTG诱导带有pGEX-4T-1-hTERT质粒的BL21细菌融合蛋白的表达，取不同时间的培养物进行SDS—PAGE分析，并进行薄层凝胶光密度扫描测定目的蛋白表达量，同时对表达产物进行Western—blot检测。结果对SDS-PAGE结果分析发现，含pGEX-4T-1-hTERT质粒的BL21细菌能够表达出63kD的融合蛋白。薄层凝胶光密度扫描测定其表达水平最高为28．4％，以IPTG诱导4h表达量最高。Western—blot检测表明该融合蛋白可被hTERT多抗识别。结论诱导pGEX-4T-1—hTERT重组质粒，获得分子量为63kD的GST-hTERT融合蛋白。用抗hTERT目的基因片段表达的相应抗原蛋白。     

MAGE-3 原核重组表达载体的构建和表达

 目的 构建原核重组表达载体pGEX-4T-1-MAGE-3并检测其在大肠杆菌（E.coli）BL21中的表达。方法 RT-PCR法制备MAGE-3目的基因,采用DNA重组技术将其克隆至pGEM-TEasy和亚克隆至pGEX-4T-1载体,转化E.coliBL21株,经IPTG诱导,12%SDS-PAGE电泳分离和Western Blot表达鉴定。结果 扩增出349bp的MAGE-3目的基因并构建了原核重组表达载体pGEX-4T-1-MAGE-3;测序结果与Gen Bank收录序列相一致;在E.coli BL21中检测到含该重组表达载体的转化菌表达出分子量约35kD的融合蛋白并证实其为目的蛋白。结论 成功构建的原核重组表达载体pGEX-4T-1-MAGE-3及其所表达的融合蛋白,为以MAGE-3为基础的肽疫苗及特异诊断试剂的研制提供抗原打下了基础,为后续实验提供了依据。     

可溶性FLT-1基因的克隆以及原核表达

目的:克隆可溶性血管内皮生长因子受体1(soluble vascular endothelial growth factor receptor1,sVEGFR1或sFLT-1)基因的第1~3个Ig样区域,并在原核表达系统中表达。方法:采用RT-PCR的方法从人脐静脉内皮细胞中扩增sFLT-1基因的第1~3个Ig样区域,并克隆到PMD-18T载体中,经酶切及测序证实。然后采用BamHI/EcoRI双酶切,亚克隆到原核表达载体pGEX-4T-2中,并转化至E·coliBL21菌株。应用IPTG进行诱导表达,对表达产物进行Western Blot分析。结果:经RT-PCR成功扩增了sFLT-1基因第1~3个Ig样区域;成功地克隆了sFLT-1基因第1~3个Ig样区域;经IPTG诱导的重组质粒pGEX-sFLT表达出相对分子质量约为62×103的融合蛋白,与预期的结果相符。结论:成功克隆sFLT-1基因第1~3个Ig样区域,并在E·coliBL21中表达出GST-sFLT融合蛋白,为进一步研究sFLT-1蛋白在肿瘤治疗中的作用奠定了技术基础。     

hTERT原核重组质粒的表达

目的本课题拟观察原核重组质粒pGEX4T1hTERT在BL21细菌中的表达,为进一步探讨以hTERT为靶点的肿瘤基因免疫治疗提供实验依据。方法IPTG诱导带有pGEX4T1hTERT质粒的BL21细菌融合蛋白的表达,取不同时间的培养物进行SDSPAGE分析,并进行薄层凝胶光密度扫描测定目的蛋白表达量,同时对表达产物进行Westernblot检测。结果对SDSPAGE结果分析发现,含pGEX4T1hTERT质粒的BL21细菌能够表达出63kD的融合蛋白。薄层凝胶光密度扫描测定其表达水平最高为28.4%,以IPTG诱导4h表达量最高。Westernblot检测表明该融合蛋白可被hTERT多抗识别。结论诱导pGEX4T1hTERT重组质粒,获得分子量为63kD的GSThTERT融合蛋白。用抗hTERT目的基因片段表达的相应抗原蛋白。     

Monoclonal antibodies against avian selenoprotein W

The Sel-W gene encoding avian selenoprotein W was cloned into the vector pGEX-6p-1 with GST tag. The recombinant protein Sel-W was expressed in Escherichia coli BL21 (DE3) after induction with 1 mM IPTG. Female 10-week-old BALB/c mice were immunized with the purified Sel-W recombinant protein emulsified in Freund's adjuvant. Two monoclonal antibodies against the Sel-W, 1B8 and 4H5, were produced by lymphocyte hybridoma technique. Subtypes of the MAbs 1B8 and 4H5 were all IgM, and their ascitic fluid titers were 1:2800 and 6400, respectively. In specificity, the MAbs 1B8 and 4H5 showed positive reaction with the recombinant Sel-W protein with GST tag and the synthesized Sel-W protein, and could not react with the GST tag and the recombinant Sel-N protein. In sensitivity, the detection limits of the MAbs 1B8 and 4H5 for the recombinant Sel-W protein and the synthesized Sel-W protein were 39 ng/mL and 52 ng/mL, respectively. These data suggest that the MAb 1B8 and 4H5 will have a potential use for detection and function analysis of the avian Sel-W.     

Expression of nitroreductase gene NOR<Subscript>1</Subscript> in E.Coli and the preparation of antiserum

Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyelonai antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transeription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases.The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunobiot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsuifate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography.The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.     

Preparation and characterization of polyclonal antibody against severe acute respiratory syndrome-associated coronavirus spike protein

A truncated gene (designated S1) encoding the receptor-binding domain (RBD) in the spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was amplified by PCR. The gene was cloned into prokaryotic expression vector pGEX-6P-1, resulting in a recombinant plasmid pGEX-SARS-S1. Subsequently, pGEX-SARS-S1 was transformed into host cells BL21(DE3)pLysS, and the expression of the S1 protein was induced by isopropyl β-D-thiogalactoside (IPTG). Polyclonal antibody against SARS-CoV S1 protein was generated in a rabbit immunized with the purified S1 protein. The reactivity of the antibody to the SARS-CoV S1 protein was confirmed by Western blot analysis. ELISA indicated that the antibody against SARS-CoV S1 protein had no cross reaction with S1 proteins of transmissible gastroenteritis virus, a porcine coronavirus, and infectious bronchitis virus, an avian coronavirus. The SARS-CoV S1 protein and its antibody are valuable reagents for related studies.