溶藻弧菌热休克蛋白70基因真核表达载体的构建及鉴定

目的构建溶藻弧菌热休克蛋白70(HSP70)真核表达载体pcDNA3.1。方法提取溶藻弧菌基因组DNA,PCR扩增HSP70基因片段,克隆至TA载体,通过PCR、酶切及测序鉴定后,将HSP70基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.1,构建pcDNA3.1重组质粒,通过PCR、酶切及序列分析对HSP70基因pcDNA3.1重组质粒进行鉴定。结果扩增出1 914bp的溶藻弧菌HSP70基因片段,并成功构建溶藻弧菌HSP70真核表达载体pcDNA3.1。结论成功构建了溶藻弧菌HSP70真核表达载体pcDNA3.1,为HSP70基因疫苗研制奠定了基础。     

一个包含HCV IRES的高效真核双顺反子表达载体的构建

目的利用丙型肝炎病毒（HCV）的内部核糖体进入位点（IRES）元件构建一个新的真核双顺反子表达载体。方法克隆包含HCV5’非编码区18nt开始到HCV CORE区编码基因的32nt的IRES序列替换商用载体pIRES中脑心肌炎病毒（EMCV）的IRES序列。构建真核双顺反子表达载体pCVIR，再将绿色荧光蛋白（GFP）和乙型肝炎病毒表面抗原（HBsAg）基因分别克隆到pIRES和pCVIR中的IRES的上下游，用流式细胞仪检测GFP的表达强度，ELISA法检测HBsAg的表达，比较两者的表达效率。结果pCVIR载体对IRES下游GFP和HBsAg的表达效率高于pIRES载体。结论构建了一个高效的真核双顺反子表达载体。     

人肺新型抗蛋白酶性保护因子elafin cDNA的克隆、鉴定及真核表达载体的构建

目的克隆人肺新型抗蛋白酶性保护因子elafin基因cDNA，构建该基因的真核高效表达载体。方法抽提人肺总RNA进行RT-PCR法扩增，产物由pUCm-T载体装载、DNA测序确定后，用双酶切将Elafin cDNA定向克隆到绿色荧光蛋白表达载体pEGFP-N1，转化于大肠杆菌DH5α，筛选阳性克隆，双酶切鉴定重组质粒。结果经RT-PCR获得400b的产物，经DNA测序，确定所扩增片段为人elafin基因cDNA，进而成功筛选出真核表达载体pEGFP-N1-elafin。结论成功构建人elafin基因cDNA真核表达载体，为进一步深入研究慢性阻塞性肺疾病的发病机制奠定了技术基础。     

弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4的构建

目的构建弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4。方法根据弓形虫RH标准株SAG1和SAG4基因序列分别设计一对特异引物(分别含有HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ内切酶位点),PCR扩增目的基因,并将这两段基因分别克隆至PEGM-T Easy载体,经菌落PCR和双酶切鉴定;用HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ分别双酶切PEGM-T Easy-SAG1、PEGM-T Easy-SAG4和pVAX1,得到含内切酶位点的SAG1和SAG4基因片段,分别与pVAX1连接,构建pVAX1-SAG1和pVAX1-SAG4真核表达质粒,经菌落PCR和双酶切鉴定。结果PCR扩增得到目的基因SAG1和SAG4,测序结果分别与弓形虫RH标准株SAG1和SAG4基因比较,符合率均为100%。构建PEGM-T Easy-SAG1、PEGM-T Easy-SAG4克隆质粒和pVAX1-SAG1、pVAX1-SAG4真核表达质粒,分别经菌落PCR和双酶切鉴定,显示722bp的SAG1基因片段和511bp的SAG4基因片段均插入载体PEGM-T Easy和pVAX1中。结论成功克隆了pVAX1-SAG1、pVAX1-SAG4真核表达质粒,为弓形虫基因疫苗应用研究奠定了基础。     

携带增强绿色荧光蛋白基因的TSLC1真核表达载体构建及鉴定

目的构建携带增强绿色荧光蛋白基因的TSLC1真核表达载体,为研究其抗肿瘤功能奠定基础。方法提取基因组RNA,利用特异性引物进行RT-PCR反应,扩增全长TSLC1基因片段,与真核表达载体pReceiver-M29载体进行连接,并转化到大肠杆菌JM109中扩增,以获得重组载体,应用酶切、PCR及测序鉴定此重组载体。结果RT-PCR获得约1 329 bp大小的特异性TSLC1基因片段;测序鉴定证实,TSLC1基因片段正确插入真核表达载体pReceiver-M29中。结论成功构建了真核表达载体pReceiver-M29-TSLC1。     

大鼠TIMP-1真核表达载体的构建及其在CHO细胞中的表达

目的 构建大鼠基质金属蛋白酶组织抑制物-1(TIMP-1)的真核表达载体pTIMP-1,并在中国仓鼠卵母(CHO)细胞中表达.方法 从大鼠肝组织提取总RNA,RT-PCR扩增出TIMP-1基因的cDNA片段,将目的 片断克隆至pMD18-T载体,酶切和测序鉴定,然后将目的 片断酶切回收后插入pcDNA3.1,构建真核表达载体pTIMP-1,将该载体转染到CHO细胞后24～48 h,收集细胞上清同时制作细胞爬片,双抗体夹心酶联免疫吸附法(ELISA)和免疫组化检测TIMP-1的表达情况.结果 扩增出了长约654 bp的基因片段,并将其克隆至pMD18-T载体,酶切和测序鉴定无误;将pMD-TIMP-1中TIMP-1基因亚克隆至pcDNA3.1载体,酶切鉴定无误;pTIMP-1载体转染CHO细胞后,ELISA法可检测到细胞上清中有大量TIMP-1的表达,同时细胞爬片免疫组织化学染色可见胞浆中存在明显的阳性棕染.结论 成功构建了大鼠TIMP-1基因的真核表达载体pTIMP-1,为进一步探讨其功能奠定了实验基础.     

四环素基因表达调控系统调控HSV-tk基因表达的单载体构建

目的 构建能够调控自杀基因表达的单载体四环素基因表达调控系统.方法 以真核表达载体pcDNA3.1为载体构建骨架,将两个四环素基因表达调控元件(TetO2)克隆到pcDNA3.1载体CMV启动子下游,构建成pcDNA3.1-TetO2载体;将单纯疱疹病毒胸苷激酶(HSV-TK)与核糖体进位位点(IRES)相连的基因片段克隆入pcDNA3.1-TetO2载体的多克隆酶切位点,构建成pcDNA3.1-TetO2-HSV-TK-IRES载体;将四环素调控子基因(TR)克隆至pcDNA3.1-TetO2-HSV-TK-IRES载体IRES下游,从而构建成pcDNA3.1-TetO2-HSV-TK-IRES-TR载体.利用酶切与测序鉴定克隆结果.结果 载体构建过程的酶切结果以及测序结果表明载体构建正确.结论 成功构建了能够调控自杀基因表达的单载体四环素基因表达调控系统,为调控自杀基因体外杀伤肿瘤奠定实验基础.     

IL-37b基因克隆及其真核的表达载体构建

目的 克隆IL-37b编码区基因,并构建其真核表达载体.方法 通过RT-PCR扩增IL-37b编码区基因,DNA测序鉴定后,将阳性克隆提取质粒,通过酶切连接将目的片段定向克隆至真核表达载体pcDNA 3.1,通过菌落PCR和DNA测序等进行鉴定.结果 凝胶电泳显示RT-PCR扩增出1条大小约650 bp特异条带,DNA测序结果显示获取了大小为654 bp的IL-37b编码区基因.PCR和DNA测序对所构建的真核表达载体鉴定结果表明IL-37b编码区基因正确插入真核表达载体pcDNA3.1.结论 成功克隆了IL-37b编码区基因,并成功构建其真核表达载体,为下一步IL-37b生物学作用的深入研究奠定了基础.     

乙型肝炎病毒S基因的克隆

目的:对乙型肝炎病毒S基因进行克隆。方法:依据S基因两侧序列设计引物并引入酶切位点;聚合酶链式反应(PCR)扩增目的基因;对PCR产物和载体质粒pCMV-tag2B分别作EcoRⅠ和HindⅢ双酶切后,T4连接酶连接;将连接产物转化为大肠杆菌DH5α;增菌培养后提取重组质粒;对重组质粒作PCR、酶切及测序鉴定。结果:PCR扩增出目的基因片段;目的片段与载体连接后成功;获得含重组质粒的DH5α阳性克隆;经鉴定和测序,重组质粒含有完整正确的S基因序列。结论:成功构建了S基因的克隆载体,获得大量S基因片段,为研究S基因对HBsAg检测的影响奠定了基础。     

弓形虫pcDNA3-ROP2-p30-HSP70复合基因真核表达重组质粒的构建

目的构建弓形虫pcDNA3-ROP2-p30-HSP70真核表达重组质粒。方法根据HSP70基因序列,设计合成HSP70基因的引物,PCR扩增HSP70基因片段,克隆入pMD18-T载体,再经酶切、连接等,亚克隆至pcDNA3-ROP2-p30表达重组质粒中,并进行酶切、PCR和测序鉴定。结果 PCR扩增出长度为916bp的HSP70基因片段,将其克隆到pMD18-T中,成功亚克隆获得pcDNA3-ROP2-p30-HSP70表达重组质粒。测序结果显示,pcDNA3-ROP2-p30-HSP70表达重组质粒包含了HSP70蛋白基因完整序列。结论成功构建弓形虫pcDNA3-ROP2-p30-HSP70真核表达重组质粒,为下一步核酸疫苗的研究奠定了基础。     

pEGFP?N1?HBsAg?ROP2多基因重组表达质粒的构建及表达鉴定

目的 构建pEGFP?N1?HBsAg?ROP2重组质粒,并转染HEK293T细胞进行表达鉴定,为弓形虫病核酸疫苗的研制奠定基础。 方法 根据HBsAg基因序列和pcDNA3?p30?ROP2重组质粒酶切位点设计引物,PCR扩增HBsAg基因,经酶切、连接、转化,利用HBsAg基因替换p30基因,构建pcDNA3?HBsAg?ROP2重组质粒;经HindⅢ和KpnⅠ双酶切,将HBsAg?ROP2片段与pEGFP?N1真核表达载体相连,构建pEGFP?N1?HBsAg?ROP2重组表达质粒,转染HEK293T细胞,观察其蛋白表达水平。结果 HBsAg片段PCR产物约700 bp,与理论值相符;构建pcDNA3?HBsAg?ROP2重组质粒,双酶切电泳后得到约5.4 kb和1.9 kb的两条带,与预期结果相符。pEGFP?N1?HBsAg?ROP2双酶切后产生约4.7 kb和1.9 kb的条带,经测序鉴定,与GenBank发表的序列同源性为99.84%。目的基因已成功转染入HEK293T细胞中,且正确表达,蛋白浓度为3.08 mg/ml。结论 成功构建pEGFP? N1?HBsAg?ROP2重组表达质粒,转染HEK293T细胞能正确表达。     

辅助性T细胞表位HBV表面抗原融合基因真核表达质粒的构建

为提高HBVDNA疫苗的免疫效率，将一个通用型辅助性T细胞表位基因引入HBV表面抗原基因的5‘末端，构建成真核表达质粒。PCR方法合成PADRE-HBsAg目的基因，产物与pMDl8T载体连接，经Hind Ⅲ，EcoR Ⅰ双酶切，再克隆入真核表达载体pcDNA3．1( )。酶切及测序鉴定，该真核表达载体构建成功，为进一步观察其特异性细胞和体液免疫反应奠定了基础。     

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...     

融合基因IFN-α1b/CSPⅡ原核表达载体的构建及在大肠埃希菌中的表达

目的　构建融合基因IFN α1b/CSPⅡ的原核表达载体并予以表达。　方法　采用聚合酶链反应(PCR)从人基因组DNA中扩增出IFN α1b基因 ,克隆入原核表达载体 pGEX 4T 1,构建原核表达载体 pGEX 4T 1/IFN α1b。利用PCR法从恶性疟原虫基因组DNA中扩增出环子孢子蛋白Ⅱ区 (CSPⅡ )基因 ,克隆入原核表达载体 pGEX 4T 1,构建原核表达载体pGEX 4T 1/CSPⅡ 。用限制性内切酶BamHⅠ和EcoRⅠ将IFN α1b从原核重组质粒 pGEX 4T 1/IFN α1b中切下 ,克隆入经相同酶切的原核重组质粒pGEX 4T 1/CSPⅡ 中 ,构建融合基因的原核表达载体 pGEX 4T 1/IFN α1b/CSPⅡ。融合基因IFN α1b/CSPⅡ经异丙基 β D硫代半乳糖苷 (IPTG )诱导 ,在大肠埃希菌中进行初步表达。结果　构建的原核表达载体 pGEX 4T 1/IFN α1b、pGEX 4T 1/CSPⅡ和 pGEX 4T 1/IFN α1b/CSPⅡ经PCR和酶切鉴定与预期结果一致。证实融合基因IFN α1b/CSPⅡ拼接成功并正确地克隆入原核表达载体。在大肠埃希菌中表达出融合蛋白IFN α1b/CSPⅡ ,该融合蛋白经十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)分析与理论预测值相符。经蛋白质印迹法 (Westernblotting)鉴定具有免疫原性。　结论　构建了融合基因IFN α1b/CSPⅡ的原核表达载体 ,并在大肠埃希菌中表达了。     

抗汉坦病毒核衣壳蛋白单链抗体基因的克隆与鉴定

目的将已筛选出的抗汉坦病毒核衣壳蛋白（NP）抗原单链抗体基因克隆入原核表达载体pGEX-6p-1,构建重组表达质粒pGEX-6P-1-scFv。方法提取含抗NP抗原单链抗体基因的T7噬菌体DNA,此为模板,PCR扩增单链抗体基因,BamHI和SalⅠ双酶切,经连接酶与BamHⅠ和SalⅠ双酶切的表达载体pGEX-6p-1连接;重组DNA转化入宿主菌E．coli BL-21（DE3）,琼脂糖凝胶电泳初筛选阳性重组质粒,并用PCR法、双酶切法及DNA测序鉴定。结果重组质粒pGEX-6P-1-scFv经BamHⅠ和SalⅠ双酶切,片段大小分别为5000bp和750bp,与预期值一致;重组质粒PCR产物大小为750bp,与预期值一致;测定重组质粒序列,并与scFv序列比较,结果相一致。抗NP单链抗体基因序列克隆人表达载体pGEX-6p-1。结论成功构建了含抗汉坦病毒NP抗原单链抗体基因的重组原核表达质粒pGEX-6P-1-SCFv。     

pcDNA3?HBsAg?p30?ROP2真核表达载体的构建与鉴定

目的 目的 构建pcDNA3?HBsAg?p30?ROP2多基因重组表达载体, 并对其进行初步鉴定。方法 方法 根据重组体pcDNA3? p30?ROP2酶切位点和乙型肝炎表面抗原 （HBsAg） 基因序列等因素设计合成引物, 扩增HBsAg目的基因片段, 再应用酶切、 连接等分子生物学技术将HBsAg目的基因克隆至pcDNA3?p30?ROP2表达载体中。应用聚合酶链反应 （PCR） 初筛, 再采用 酶切、 测序等技术对构建的重组表达载体pcDNA3?HBsAg?p30?ROP2进行鉴定。 结果 结果 PCR扩增出HBsAg基因片段, 构建 了pcDNA3?HBsAg?p30?ROP2多基因真核表达载体。PCR与酶切结果显示, 该基因片段大小均与理论值相符; 测序结果显 示该重组表达载体包含了p30?ROP2和HBsAg目的基因的完整序列。 结论 结论 成功构建了多基因重组表达载体pcDNA3? HBsAg?p30?ROP2, 为进一步研究多基因核酸疫苗奠定了基础。     

HBsAg真核表达质粒及其诱导的小鼠特异性免疫应答

目的 研究HBsAg真核表达质粒pCI-S和pcDNA3.1-S在真核细胞中的表达和质粒DNA的免疫效果。方法 应用基因重组技术构建HBsAg真核表达质粒pCI-S和pcDNA3.1-S；经酶切和测序鉴定无误后，用阳离子脂质体介导的方法将重组质粒转染HepG2和COS-7细胞，48h后，再ELISA的方法检测重组质粒在细胞中HBsAg的表达，同时同质粒DNA免疫小鼠，用ELISA检测免疫小鼠血清抗-HBs抗体水平；用乳酸脱氢酶释放法检测小鼠肿瘤细胞HBsAg特异性CTL反应。结果 重组质粒pCI-S和pcDNA3.1-S转染的HepG2和COS-7细胞培养上清液和sAg均为阳性。DNA免疫小鼠血清可检测到高滴度的抗-HBs抗体，免疫小鼠脾细胞可检测到校强的HBsAg特异性CTL反应。结论 HBsAg真核表达质粒pCI-S和pcDNA3.1-S可在HepG2和COS-7细胞中高效表达，DNA免疫小鼠成功地诱导出抗-HBs和HBsAg特异性CTL反应。     

Cost-effective method of siRNA preparation and its application to inhibit hepatitis B virus replication in HepG2 cells

AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture. METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase III in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2 000 to test their inhibition efficiency. RESULTS: siRNAs corresponding to part of the hepatitis B virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP. CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.     

口蹄疫病毒O/China/99株多基因植物组成型表达载体的构建及序列分析

目的构建包含FMDV结构蛋白P1、非结构蛋白2A、3C以及部分2B的基因片段P12X3C的植物组成型表达载体。为FMDV可饲疫苗的研制奠定基础。方法通过PCR扩增法，从pGEM／P12A质粒和pGEM／3C质粒中扩增P12A及部分2B基因（P12X）、3C基因，将获得的P12X与3C片段经Bam HI／SpeⅠ和SpeⅠ／SalⅠ双酶切后，与经Bam HI／SalⅠ双酶切后的pUC18质粒大片段连接，得到重组质粒pUC18／P12X3C，pUC18／P12X3C质粒经Bam HI／SalⅠ双酶切后的大片段与经同样双酶切的植物表达载体pBin438连接。转化子经酶切、PCR及测序鉴定后。获得包含FMDVO／China／99株P12X3C片段的植物组成型表达载体pBin438／P12x3C，最后通过三亲交配法，将表达载体pBin438／P12x3C导入农杆菌。结论成功构建FMDV多基因的植物组成型表达载体。     

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of α determinant region

AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice. METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal antiHBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106+26.7 vs i 814.4+776.3, P<0.01,t=5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs6/10, 4.56+3.52 mIU/mL vs27.60±17.3 mIU/mL, P=0.02, t=2.7). CONCLUSIONS: HBsAg with deleted second-loop of the α determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.