Expression of Duck Interferon Alpha in BL21（DE3）plysS

To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic ex-pression vector of DuIFN-α was constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the proteingene of DuIFN-α was cloned from pMD-18-duIFN-α recombinant. The gene was then inserted to pGEM-T vectorand identified by restriction endonuclease analysis and sequencing. DuIFN-α was ligated with the prokaryotic expres-sion vector of pET30 a, then transformed into BL21 (DE3) plysS. The best inducing time and IPTG concentration for the expression of this recombinant protein was tested through the expression of the positive recombinant with differ-ent time span and different IPTG concentration. Lots of the protein of DuIFN-α were expressed in BL21 (DE3)plysS with 1 mmol·L-1 IPTG for 4 hours and its molecular weight for 34 000.     

The Prokaryotic Expression and Bioactivity of the Recombinant Red Fire Ant Venom Allergen Sol i 4

The sting of red imported fire ant （RIFA） could cause serious allergic response in fraction of people. These allergic reactions are mainly caused by its venom, especially venom allergen Sol i 1-4. To produce large amount of RIFA venom allergen Sol i 4 for diagnosis of RIFA allergy and allergen-specific immunotherapy, the gene encoding this protein was amplified and cloned into the prokaryotic expression vector pET43, la. The recombinant plasmid was used to transform competent cells and the recombinant proteins were expressed in E. coll. SDS-PAGE and Western blotting analysis indicated that high-level expression of Sol i 4 protein was successfully achieved. Allergenic activity analysis of the recombinant allergen Sol i 4 was then performed on rabbit. The result showed that the recombinant protein obtained had significant allergenic activity. It indicated that the recombinant allergen Sol i 4 of RIFA venom was successfully expressed in E. coli, which provided foundation for further developing therapeutic and diagnosis reagents of RIFA allergy.     

Preparation of Monoclonal Antibody Against PthA-NLS and Cloning of the Relative ScFv Gene

The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR （splicing by overlap extension）, and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a（＋） vector. The later plasmid was transferred into E. coli BL21 （DE3） and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.     

猪繁殖与呼吸综合症GP5蛋白的纯化与免疫活性分析(英文)

[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological method. [Method] The recombinant expression plasmid pGEX-6P-5 was transformed into BL21 and expressed after being induced with IPTG. The solubility analysis of expression products was carried out, and then the recombinant protein was purified for SDS-PAGE identification and Western-blot analysis. Finally, the recombinant antigen was used in the immune experiment of guinea pigs. [Result] The target protein content accounted for 30% of the total cells protein content according to the chromatography scanning, and the purity of target protein after purification reached 80%. The purified protein was analyzed by Western-blot and immune experiment of guinea pigs, and the results showed that the expressed protein had good reactionogenicity and immunogenicity. [Conclusion] This study provides materials for further studies on the function between PRRSV ORF5 gene and its editing protein, which also lays a foundation for porcine reproductive and respiratory syndrome virus genetic engineering products.     

Bacterial Expression and Purification of an Active ω-Atracotoxin-Arl b from Spider Atrax robustus

The ω-atracotoxin-Arlb toxin （ω-ACTX-Arl b） is one of the arthropod-selective peptide neurotoxins from the venom of Australian funnel-web spider Atrax robustus. The gene of Arlb was synthesized and cloned into pET-32a（＋） vector to allow expression of Arlb as a fusion protein with thioredoxin and the His-tag （rTrx-Arlb） in E. coli BL21 （DE3）. The optimal condition for inducing the expression ofrTrx-Arl b was 1.0 mmol L-1 IPTG for 6 h at 28℃. The fusion protein rTrx- Arlb was expressed in soluble form and was purified effectively by HisTrap HP affinity column and rpHLPC and a final yield of purified rTrx-Arlb was 95 mg from 1 000 mL E. coli culture. The LD50 values for Mythimna separate and Tenebrio molitor were 111.66 and 11.04 ug g-1 determined by injection of the purified rTrx-Arlb. The results indicated that the recombinant Arlb protein was successfully expressed in E. coli and it was high toxicity against tested insects.     

Cloning of a novel phytase from an anaerobic rumen bacterium,Mitsuokella jalaludinii,and its expression in Escherichia coli

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, p ET32 a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 k Da by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, p H 4.5 and showed good p H stability from p H 3.5 to 5.5(78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced p H stability and metal ion effects should be taken into consideration during feed enzyme formulation.     

Construction of Antibacterial Peptide CecropinB Eukaryotic Recombinant Vector and Its Expression in Dairy Goat Mammary Gland Epithelial Cells

To investigate the expression of antibacterial peptide CecropinB cDNA in dairy goat mammary gland epithelial cells,the CecropinB gene was cloned and was inserted into a eukaryotic vector pECFP-C1 to construct the recombinant plasmid pECFP-B by genetic engineering technique.Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CecropinB.The expression of CecropinB was also detected.The result of RT-PCR demonstrated CecropinB gene was expressed in transfected cells.CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland,exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.     

金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达(英文)

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.     

家蚕浓核病毒镇江株非结构蛋白2(NS2)的表达及活性研究

 【目的】对家蚕浓核病毒镇江株非结构蛋白2（NS2）基因进行克隆、表达,并测定表达产物的生物活性。【方法】利用PCR技术从家蚕浓核病毒镇江株（BmDNV-Z）基因组中扩增得到非结构蛋白2(NS2)基因片段,将其克隆到表达载体pET28a得到重组表达质粒pET28a-NS2,在大肠杆菌BL21（DE3）中进行表达,SDS-PAGE和Western blot检测,表达产物经Ni柱纯化,经过复性检测其Helicase和ATPase活性。【结果】克隆获得了BmDNV-Z NS2基因,并在大肠杆菌中得到了成功表达,表达产物经Ni柱纯化获得了目的蛋白NS2。纯化的NS2蛋白具有Helicase活性,能将双链DNA底物解旋成为单链,并且具有一定的底物极性选择性,对于极性底物表现出更高的解旋活性。同时,纯化的NS2蛋白具有ATPase活性,其酶活力可达到0.276 μmol?μg-1?h-1。【结论】BmDNV-Z NS2基因编码的病毒非结构蛋白具有Helicase和ATPase活性,并且Helicase活性具有一定的底物极性选择性,推测该基因在病毒DNA的复制过程中发挥重要作用。     

固始鸡白细胞介素2基因的克隆、表达及其表达蛋白活性的检测

参照GenBank上已发表的鸡白细胞介素2序列设计引物,运用RT-PCR技术,从经ConA诱导的 20～35日龄固始鸡脾细胞总RNA中扩增出目的片段,IL-2基因,将其插入到pGEM-T载体上,构建了克隆质粒 pGEM-T-chIL-2。测序结果表明,本试验克隆的固始鸡白细胞介素2基因与GenBank上已发表的序列相比,存在2 个核苷酸变异。重新设计表达引物,以克隆质粒pGEM-T—chIL一2为模板PCR扩增表达片段,对表达片段进行 Hind Ⅲ和BamH Ⅰ双酶切,连接到作同样双酶切的pET28a表达载体上,鉴定后转化大肠杆菌BL21并进行IPTG 诱导表达,然后对表达产物进行SDS-PAGE电泳鉴定,结果表明,表达蛋白主要以包涵体的形式存在,表达蛋白分子质量约为18 ku;目的蛋白表达量占总量的18％。体外活性检测表明,重组蛋白具有促进淋巴细胞增殖的活性。     

猪细小病毒SY-99株非结构蛋白NSl基因的克隆与原核表达

根据猪细小病毒 (porcineparvovirus ,PPV)NADL 2株非结构蛋白基因NS1序列设计引物 ,扩增出了PPVSY 99株的NS1基因 ,将其克隆到原核表达载体pET2 8a中 ,得到重组质粒SYNS1/pET2 8a。将SYNS1/pET2 8a转化到大肠杆菌BL2 1(DE3)中 ,经IPTG诱导 ,使PPVNS1基因获得了表达 ,运用ELISA和Westernblotting证实了表达产物的特异性。经表达产物细胞定位分析 ,表达的NS1蛋白是以包涵体的形式存在于大肠杆菌中。通过改变诱导时间或诱导剂IPTG的浓度 ,确定了表达NS1蛋白的最佳诱导条件 :IPTG终浓度为 0 .6mmol·L-1,诱导时间为 3h ,在大肠杆菌中最高表达含量为 17.8%。亲和层析后 ,得到了纯化的NS1蛋白 ,在Westernblotting检测中 ,纯化蛋白大大降低了反应背景。表达的NS1蛋白可作为免疫诊断试剂来检测PPV的发生 ,而且还可用于鉴别灭活疫苗免疫猪和自然感染猪     

黄单胞杆菌纤维素酶的原核表达及固定化研究

从野油菜黄单胞杆菌(Xanthomonas campestris pv.Campestris)中PCR克隆得到删除信号肽的纤维素酶基因engXCAΔSP,并成功使用CPEC(circular polymerase extension cloning)方法构建了原核表达载体pET28a-engXCAΔSP;将该载体转入大肠杆菌rosetta(DE3)中,用IPTG诱导蛋白质的表达;通过Ni-NTA树脂非变性亲和纯化到了较纯的蛋白。SDS-PAGE电泳结果表明:engXCAΔSP基因编码出约50 kD的蛋白质ENGXCAΔSP。然后对该酶成功进行了固定化。该酶比活为60 U.mg-1,固定前后最适温度为53℃与62℃,最适pH为5.4与5.8,动力学常数分别为Vmax:411μmol.mL-1.h-1与383μmol.mL-1.h-1,Km:0.2500%与0.3125%。     

短小芽胞杆菌抑菌蛋白TasA基因克隆及功能分析

为了研究短小芽胞杆菌Bacillus pumilus DX01菌株的抑菌相关基因的功能,采用PCR方法从DX01基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建原核表达载体pET2-TasA,在大肠杆菌Escherichia coli BL21中表达获得TasA基因的融合表达蛋白,分别利用悬滴法和平板打饼法检测融合蛋白对玉米小斑病菌(Bipolaris maydis)分生孢子萌发和对玉米小斑病菌及水稻稻瘟病菌菌丝体生长的抑制活性,结果表明TasA融合蛋白对这2种植物病原真菌的生长有着显著的抑制作用。TasA基因包含1个780bp的完整开放阅读框(GenBank:KC692521),编码259个氨基酸残基;该序列与B.subtilis菌株PEBS2501(GenBank::FJ713581)、B.amyloliquefaciens HF-01(GenBank:JF781312)和B.subtilis菌株BWST7003(GenBank:AP012496)同源抑菌蛋白基因序列相似性达99%。原核表达产物经SDS-PAGE分析检测到约32.3kDa的融合蛋白;经Ni-NTA柱纯化后的融合蛋白对供试病原菌均有显著的抑制作用。经测定发酵液的TasA融合蛋白产率为28μg/mL,短小芽胞杆菌抑菌蛋白TasA基因具有良好的抑菌效果。     

Molecular Cloning and Prokaryotic Expression of Non-Structural Protein NS1 Gene of Porcine Parvovirus

Porcine parvovirus (PPV) is one of the major agents causing swine reproductive failure. NS1protein is a non-structural protein of PPV and can be used as a reagent for differentiation of vaccinated ani-mals and infected ones. In present study, a recombinant plasmid pET28a/NS1 was constructed by cloning thecoding sequence for NS1 of PPV into pET28a, a bacterial expression vector. The NS1 protein was expressed inE. coli BL21 (DE3) after induced by IPTG and the recombinant fusion protein was purified with affinity chro-matography. Expression amount of NS1 protein was improved by optimizing the inducing parameters. The re-combinant NS1 protein is reactive to PPV positive sera in Western blot and ELISA test and therefore can beapplicable in differential diagnosis of PPV infections.     

口蹄疫3D蛋白N端T细胞表位重组蛋白的表达、纯化及鉴定

为获得具有抗原性的口蹄疫病毒(FMDV)3D蛋白N端T细胞表位的重组表达蛋白,根据FMDV3D蛋白前115位氨基酸序列,设计优化并人工合成相应的核酸序列,将其克隆至pET-28a中,构建原核表达质粒pET28a-115,并将重组质粒转化至BL21(DE3)细胞。经IPTG诱导后,收集菌液进行SDS-PAGE和Western-bolt鉴定,结果显示,在16kD处有一条明显的蛋白表达条带。对表达产物进行可溶性分析,结果表达蛋白50%为可溶性蛋白,经Ni-NTA亲和层析纯化后获得了较高浓度和纯度的目的蛋白。研究结果表明,含FMDV 3D蛋白前115位氨基酸的重组蛋白在大肠杆菌中得到了高效可溶性表达,并具有较好的抗原活性,为开发口蹄疫诊断试剂和基因工程疫苗奠定了基础。     

日本脑炎病毒WHe株NS1基因的克隆、测序及表达

以日本脑炎病毒(Japanese encephalitis virus,JEV)WHe株的基因组RNA为模板,采用RT-PCR技术,克隆了JEV WHe株的NS1基因,并对其进行了测序和序列分析。构建了pET28b-NS1表达载体,转化表达宿主大肠杆菌BL21(DE3),并对其进行了诱导表达,对表达产物进行检测。结果表明,NS1基因全长1 145 bp,其核酸序列与JEV P3株同源性为99．4％,与SA14和SA14-14-2等27个JEV毒株的核苷酸序列同源性为98％,表明NS1基因的保守性很高;pET28b-NS1表达产物的相对分子质量约为43 ku,大小与预期结果相符。NS1基因克隆表达成功。     

牛病毒性腹泻黏膜病毒E2基因的原核表达与免疫原性分析

[目的]原核表达牛病毒性腹泻黏膜病毒(BVDV)E2基因编码蛋白。[方法]采用PCR方法从BVDV中扩增E2基因片段,与原核表达载体pET-32a连接,构建重组表达质粒pET-32a-E2,转化E.coli(Rosetta)感受态细胞,重组菌用1 mmol/L IPTG诱导表达E2蛋白,进行SDS-PAGE电泳,并用Ni-NTA亲和层析柱纯化目的蛋白,经Western blot分析鉴定免疫原性。[结果]重组质粒pET-32aE2经PCR及酶切鉴定证明构建正确,重组质粒能够在大肠杆菌中大量表达,表达产物的分子质量大小约为58 kDa,纯化后E2重组蛋白浓度0.521 mg/mL,Western blot分析表明,其能被BVDV阳性血清识别,具有很好的免疫原性。[结论]E2蛋白成功表达,为后续建立BVDV检测方法奠定了基础。     

哈维氏弧菌硫氧还蛋白还原酶在大肠杆菌中的表达及对大菱鲆的免疫保护作用

从致病性哈维氏弧菌Vibrio harveyi SF1基因组中扩增获得含硫氧还蛋白还原酶（trxR）基因的1 133bp目的片段,连接至载体pMD19-T Simple。测序结果表明,目的片段含有960 bp trxR基因阅读框,编码为319个氨基酸残基的蛋白质。Blast分析结果显示,硫氧还蛋白还原酶蛋白氨基酸序列与哈维氏弧菌、溶藻胶弧菌、副溶血弧菌、灿烂弧菌、弗氏弧菌的硫氧还蛋白还原酶蛋白序列的相似性分别为99%、98%、96%、94%、92%。构建表达质粒pET-28a（＋）/TrxR后转化至大肠杆菌E.coli BL21（DE3）中,用IPTG诱导表达,SDS-PAGE分析显示,重组蛋白的相对分子质量为34 000。用Ni琼脂糖亲和层析柱分离得到纯化的重组蛋白,将该蛋白以50μg/尾的剂量肌肉注射免疫大菱鲆Scophthalmus maximus,用ELISA法检测免疫1～4周内鱼血清中特异性抗体的效价。结果表明,特异性抗体效价持续升高,第2周则达到1∶128。免疫4周后用哈维氏弧菌SF1对大菱鲆进行人工感染,对照组鱼的死亡率为100%,免疫组鱼的死亡率为25%,相对免疫保护力为75%。试验表明,哈维氏弧菌TrxR蛋白具有较好的免疫原性,可作为潜在的亚单位疫苗用于哈维氏弧菌病的免疫预防。