Cloning of human sperm protein(SP17) and expression in escherichia coli DH5α

AIM: To obtain GST fusion protein of hSP17 gene and construct the recombinant plasmidfor expression in E.coli.METHODS:Total fragment of hS P17 cDNA gene were amplified by RT-PCR,then subcoloned into p GEX-3b to generate recombinant hS P17/pGEX.Right orientation of insert are identified by restricted enzyme digestion.Transform the correct recombinant plasmid into the E.coli DH5a.The expression of fusion proteins hS P17-GST were induced by adding isopropylthiogalactoside(IPTG).RESUL TS and CONCLUSION:The recombinant plasmid hS P17/pGEX-3b could express effectively in E.coli and a high level of fusion protein hsp17-GST with the predicted molecular weight was detected.     

Expression of recombinant quadrivalent Aβ1-15 and its immunogenicity identification

AIM:To prepare the second-generation Alzheimer disease vaccine of quadrivalent tandem Aβ1-15 and to explore its immunogenicity.METHODS: The 4×Aβ15 gene was amplified by PCR with the recombinant plasmid pcDNA-4×Aβ15 as a template, which was prepared by our research group before. The 4×Aβ15 gene was then cloned to the pGEX-4T-2 plasmid. The fused GST-4×Aβ15 protein was expressed when the bacteria containing the recombinant plasmid was induced. The purified 4×Aβ15 protein was then obtained after the GST-4×Aβ15 protein was digested by thrombin. Meanwhile, BALB/c mice were used to confirm whether they could generate Aβ specific humoral immunity when immunized with the 4×Aβ15 protein.RESULTS: After identification and DNA sequence analysis, the recombinant plasmid pGEX-4×Aβ15 was constructed. Expression of recombinant 35 kD GST-4×Aβ15 was verified by SDS-PAGE and Western blotting. The 9 kD 4×Aβ15 protein was produced after thrombin digestion of GST-4×Aβ15. Immunizing mice with the 4×Aβ15 protein have induced high titre anti-Aβ antibodies.CONCLUSION:The recombinant prokaryotic vector pGEX-4×Aβ15 was constructed successfully, and the purified 4×Aβ15 protein was obtained after prokaryotic expression. It is demonstrated that the 4×Aβ15 protein is a strong immunogen, confirmed by immunization of BALB/c mice.     

Prokaryotic expression and preparation of GST/BFRF3 fusion protein of Epstein-Barr virus for screening nasopharyngeal carcinomas

AIM: To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsid antigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS: DNA extracted from the B95-8 cells was used as the templates. Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene, and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The expression of GST/BFRF3 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified by glutathione-sepharose beads. The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS: The GST/BFRF3 fusion protein was successfully expressed in E. coli. The molecular weight of the product was approximately 44 kD. The recombinant fusion protein GST/BFRF3 showed good immunoreactivity. A novel ELISA was established using GST/BFRF3 protein. Serum samples collected from the NPC patients and healthy controls were tested by this ELISA. The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65％ and 87％, respectively.CONCLUSION: The recombinant protein GST/BFRF3 is expressed in E.coli, and it has diagnostic value for screening of NPC patients.     

瓜类褪绿黄化病毒p22 基因在大肠杆菌中的表达及抗血清的制备

 以被瓜类褪绿黄化病毒（Cucurbit chlorotic yellows virus,CCYV）侵染的甜瓜叶片为供试材 料,采用RT-PCR 方法克隆其P22 蛋白基因,并将其连接到原核表达载体pGex-4T-3 上,PCR 验证及克隆 测序确定开放阅读框的正确性。将重组载体pGexp22 转化大肠杆菌BL21 菌株,诱导表达,SDS-PAGE 分 析表明,经IPTG 诱导,p22 基因在大肠杆菌BL21 中得到了高效表达。以表达的蛋白作为抗原,免疫家 兔,制备了CCYV P22 的特异性抗血清。ACP-ELISA 检测结果表明,血清效价高达1.28 × 10⁵。Western blot 检测甜瓜叶片,结果表明抗血清能够特异性地检测CCYV 侵染的甜瓜叶片中的CCYV P22 蛋白。     

香蕉果实转录因子MaWRKY1基因的原核表达和多克隆抗体制备

MaWRKY1是从香蕉果实中克隆出的一个转录因子基因。为进一步研究该基因的功能,制备了MaWRKY1多克隆抗体。选取MaWRKY1基因全长中N端第168 ~ 400个氨基酸之间的包括两个WRKYGQK保守域的cDNA序列,构建了原核表达载体pET-MaWRKY1,并转化到大肠杆菌BL21中诱导表达菌体蛋白。SDS-PAGE电泳检测结果表明,His-MaWRKY1融合蛋白成功获得了高效表达,分子量在26 kD左右。His-MaWRKY1融合蛋白经过Ni-NTA琼脂糖凝胶树脂纯化,SDS-PAGE制备胶割胶富集,电洗脱法纯化后得到的纯化蛋白浓度达到0.5 mg • mL-1。经对新西兰兔进行5次免疫,获得了多克隆抗血清,采用免疫吸附方法对抗血清进行了纯化。将纯化后的抗体通过间接酶联免疫（ELISA）和蛋白质印迹（Western blot）分析,表明所制备的抗体具有很好的效价,效价比为1︰160 000,同时具有良好的灵敏度和特异性。进一步提取香蕉不同组织总蛋白,Western blot检测显示,在分子量26 kD左右处出现特异的蛋白质条带,证明所制备的抗体可以与香蕉WRKY蛋白特异性结合,并且低温可以诱导香蕉果实中MaWRKY1蛋白表达,暗示MaWRKY1蛋白表达可能与果实耐冷性有一定的关系。     

黄瓜脂氧合酶基因CsLOX2 的原核表达载体 的构建及表达分析

以先前克隆到的黄瓜脂氧合酶基因CsLOX2 全长的cDNA 为模板,用含有特异性酶切位点
的Yh1、Yh2 为引物,通过PCR 方法将黄瓜脂氧合酶基因CsLOX2 的ORF 区构建到大肠杆菌表达载体
pGEX-4T-1 上获得原核表达载体p4t-LOX2,将该表达载体p4t-LOX2 转化到大肠杆菌菌株BL21（DE3）
中,获得相应的重组工程菌。在IPTG 诱导下,通过SDS-PAGE 电泳,得到一条约115 kD 的融合蛋白条带,
除去pGEX-4T-1 自身诱导的约26 kD 大小的GST 标签蛋白后,CsLOX2 编码一个约89 kD 的蛋白。 经过
融合蛋白表达体系的优化分析,表明该融合蛋白在 37℃,0.80 mmol·L^-1 IPTG 诱导表达 10.5 h,可获得
融合蛋白的最大表达量。     

Interaction between human augmenter of liver regeneration and Na+-K+-ATPase in vitro

AIM: To check the physical interaction between GST- Na+-K+-ATPase domain and recombinant human augmenter of liver regeneration (rhALR) by GST pull down assay. METHODS: With PCR and genetic recombinant techniques, the coding region of β subunit of Na+-K+-ATPase was cloned into expressing plasmid pGEX-4T and identified by endonuclease digestion and sequencing methods. Under the inducing of 0.1 mmol/L IPTG, the fusion protein GST- Na+-K+-ATPase domain was highly expressed by E.coli DH-5α. After hypersound quassating, the GST- Na+-K+-ATPase domain was purified by glutathione agarose beads and the physical interaction with rhALR was checked by GST pull down assay. RESULTS: Analysis by SDS-PAGE showed the rhALRs of monomer and dimmer in GST- Na+-K+-ATPase domain lane. The Western blotting of the GST-pull down assay showed the same results as well. CONCLUSION: The Na+-K+-ATPase domain is associated with rhALR specifically in vitro.     

百合无症病毒16kD基因的克隆、原核表达及抗血清制备

以感染百合无症病毒(LSV)的百合叶片为试材,克隆LSV16 k D基因,连接到原核表达载体p ET-28a(+)上。将获得的重组质粒p ET-28a(+)+16 k D转化大肠杆菌BL21(DE3),经IPTG诱导得到了高效表达的16 k D蛋白,融合蛋白分子量约为20 k D。融合蛋白经过镍柱纯化后作为抗原免疫注射小鼠,制备得到16 k D蛋白抗血清。Western blot分析显示所制备的抗血清与诱导表达的融合蛋白发生特异性反应;通过ELISA检测和RT-PCR检测百合样品,证实制备的抗血清与LSV侵染的百合叶片发生了相同的特异性反应。结果表明,目的蛋白表达成功,所制备的抗血清具有特异性,可用于LSV的快速检测、免疫组织化学以及16 k D蛋白功能研究。     

柑橘溃疡病致病基因PthA核定位信号肽抗血清制备及其抑菌的研究

 用PCR法扩增位于柑橘溃疡病菌致病基因pthA C-末端的3个核定位信号序列,并将其克隆到原核表达载体PET32a(+)上,经双酶切及核酸序列测定重组质粒(PthA-NLS),其序列与GenBank中pthA的相关序列有99.9%的同一性。重组质粒转化大肠杆菌BL21(DE3)后诱导了重组多肽的表达,并用Ni2+-NTA纯化柱得到了48kD的纯化重组多肽。把重组多肽注入免疫Balb/c小白鼠,制备了相应的抗血清,Western Blotting和ELISA分析结果表明,抗血清可特异地结合重组多肽,亦可识别溃疡病菌PthA天然蛋白,获得的抗血清可以用于柑橘溃疡病的检测。利用抗血清与溃疡病菌混合接种离体冰糖橙叶片,发现抗血清能推迟溃疡病菌的致病过程,且病斑比对照小,但未能达到抗病的程度。pthA基因末端核定位信号序列的克隆、原核表达及抗血清的制备为进一步研究pthA的致病机理和研发溃疡病快速分子检测技术奠定了基础。     

High efficient expression of human endostatin in E.coli and its antiangiogensis activity

AIM: To examine the expression of human endostatin in E.coli, produce its fusion protein antibody and observe its biological activity. METHODS: Endostatin gene was amplified by polymerase chain reaction,recombined with plasmid vector pGEX-2T and induced expression with IPTG.The protein activity was tested by endothelial cell proliferation inhibitory assay.Inclusion body crudely purified was used to generate polyclonal antibody to detect its expression at mouse's liver and kidney etc. RESULTS: The protein expressed was 20kD after digestion by thrombin,it appeared the anti-angiogenesis activity and Western blotting indicated the expression of endostatin in liver and kidney of mouse. CONCLUSION: The successful expression of human endostatin and the preparation of polycolonal antibody indicated its potential application in anti-angiogenesis therapy and diagnosis tumors.     

Preparation and characterization of anti-hBLyS monoclonal antibody by DNA immunization

AIM:To establish the monoclonal antibody against human B lymphocyte stimulator (hBLyS) by DNA immunization and analyse its characterization. METHODS:The 858 bp DNA fragment of hBLyS was cloned into pcDNA3 plasmids. The cloned insert was identified by both sequence analysis and double digestion of the recombinant plasmid with restriction enzymesXho Iand EcoR I. After the splenocytes from BALB/c mice immunized with the recombinant plasmid of pcDNA3/hBLyS were fused with myeloma cells SP2/0,the hybridoma which can produce monoclonal antibodies against hBLyS were obtained. The specificity of anti-BLyS monoclonal antibody from hybridoma was verified by ELISA, Western blot and flow cytometry. RESULTS:The recombinant mammalian cell expression vector of pcDNA3/hBLyS was constructed,the sequence of the insert gene was identified to be the sequence encoding hBLy S antigen. The culture supernatants of hybridoma 9c10 were tested to be the monoclonal antibody with specificity against hBLyS on human peripheral blood CD3⁺T cell activated by hIFN-γ by ELISA,Western blot and flow cytometry.CONCLUSION:The monoclonal antibodies against hBLyS with high activity and specificity have been established successfully, and will be an useful tool in the studies of relationship between hBLyS and human autoimmunity diseases.     

香蕉线条病毒衣壳蛋白功能域基因的原核表达及抗血清制备

 制备香蕉线条病毒广东分离物（BSV-GD）的抗血清,可为BSV-GD 的快速检测和进一步研 究BSV 编码蛋白的功能提供条件。通过常规分子生物学方法,克隆了BSV-GD 衣壳蛋白（Coat protein, CP）功能域基因,构建了该基因的原核表达载体pET28b-CP,并诱导表达了大小约为46.5 kD 的融合蛋 白His-CP。可溶性分析表明该融合蛋白以包涵体形式存在。利用His 标签纯化试剂盒对目的蛋白进行纯 化、回收,获得了高纯度的融合蛋白。以纯化的融合蛋白为抗原免疫健康大耳白兔,成功制备了BSV-GD CP 功能域基因编码蛋白的兔抗血清。Western-blotting 分析表明该抗血清具有很强的特异性,间接ELISA 法检测抗血清效价达204 800 倍以上,对植物材料的合适检测浓度为1︰1 600 ~ 1︰6 400。     

温州蜜柑萎缩病毒小外壳蛋白基因克隆与原核表达及其抗体制备

 用RT-PCR方法,从采自重庆奉节的‘宫本’柑橘的2个样品中扩增出温州蜜柑萎缩病毒（Satsuma dwarf virus,SDV）SDV RNA2的3′末端,长度为975 bp,与报道的SDV S-58 3′末端序列同源性分别为98.7%和98.4%。根据获得的序列设计引物,以含有SDV FJ 3′末端序列的质粒为模板,PCR扩增获得大小为 654 bp的SDV-FJ小外壳蛋白（Small coat protein,CPS）基因产物。构建了CPS与GST融合表达载体PGEX-CPS,在37 ℃、1.0 mmol · L-1 IPTG条件下诱导表达,SDS-PAGE电泳分析表明成功表达出分子量约为42 kD的GST-CP融合蛋白。以表达的融合蛋白为抗原免疫家兔,制备的抗血清的效价为1/12 800。用获得的抗体进行组织印迹分析表明,SDV在柑橘叶柄基部韧皮部维管束区域含量最高。     

平榛脱水素基因的克隆与表达分析

 以平榛（Corylus heterophylla Fisch.）花芽为试材,采用RT-PCR和RACE方法克隆了一个平榛与脱水素基因同源的cDNA基因,命名为ChDHN（GenBank登录号HM228389）,其全长639 bp,具有一个504 bp的潜在编码区,编码167个氨基酸组成的多肽,具有LEA类家族成员具有的特征多肽序列,属于Y₄SK₂类型DHN基因,预测ChDHN蛋白质分子量18.03 kD,预测其理论等电点为7.28。对ChDHN的时空表达特性进行了研究,以Actin为内参,对ChDHN在4 ℃冷激条件下（0、2、4、8、24和48 h）的表达模式进行了初步的研究,冷激处理后ChDHN表现逐渐上调的表达趋势,24 h达到最大表达量,48 h表达量降低;推测ChDHN属于植物冷适应调节网络中的应答基因;定量RT-PCR分析ChDHN在不同器官中的表达,在种子中高丰度表达,其次是雄花序和花芽,在树皮中表达最低。用PCR、酶切和测序鉴定等方法检测已成功构建重组表达载体pET-32a(+)-DHN,将鉴定完全正确的重组质粒转化大肠杆菌BL21(DE3),经SDS-PAGE分析并经过Western blotting鉴定,表明重组蛋白被IPTG诱导后高效表达出一条比预测分子量18.03 kD大4 kD的融合蛋白。     

甜樱桃DELLA蛋白基因PaGAI的克隆与表达分析

 利用RT-PCR和RACE技术从甜樱桃（Prunus avium L.）嫩叶中克隆了赤霉素信号转导途径中的关键负调控因子DELLA蛋白基因cDNA全序列,将其命名为PaGAI。该基因cDNA全长2 310 bp,开放阅读框ORF为1 788 bp,推测其编码一个含有595个氨基酸残基的多肽链,蛋白质分子量约64 kD。生物信息学分析结果表明,PaGAI编码蛋白具有DELLA蛋白保守结构域,其N端存在两个非常保守的酸性结构域DELLA和VHYNP作为信号感知区域,C端有VHIID、RVER 和SAW结构域作为阻遏区域。PaGAI与其它植物的DELLA蛋白具有较高的同源性,其中与苹果MdRGL2b蛋白同源性最高,达到84%。构建pGEX-4T-1/PaGAI原核表达体系,并转化E. coli BL21,经0.5 mmol · L-1 IPTG诱导蛋白表达,SDS-PAGE检测获得了分子质量为91 kD的融合蛋白,半定量RT-PCR分析表明,PaGAI在花、果实、叶片、韧皮部中普遍表达,在花与韧皮部中的表达远远强于在果实与叶片中的表达。     

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。     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Expression features of human decorin in E.coli DH5α

AIM: To study the expression features of human decorin in E. coli DH5α.METHODS: The pGEX-4T-1-decorin fusion clone was expressed in E. coli DH5α. Positively expressed clone was selected by SDS-PAGE. The optimized inducing time by 1 mmol/L IPTG was determined. The solubility of GST-decorin fusion protein was analyzed by ultrasonic crush method, and its quality was examined by Western blotting. RESULTS: With the induction of 1 mmol/L isopropy-β-D-thiogalactoside (IPTG), the fusion protein was expressed in E. coli DH5α. The optimized inducing time by 1 mmol/L IPTG was 4 hours. Most of fusion protein existed in the form of inclusion body. The expressed protein was GST fusion protein.CONCLUSION: It is suggested that the fusion protein of GST-decorin may be expressed in E.coli DH5α in a large amount in the form of inclusion body.