结核分枝杆菌Ag85A基因DNA疫苗的构建和免疫原性研究

目的　构建编码结核分枝杆菌蛋白Ag85A基因为基础的DNA疫苗。 方法　采用聚合酶链反应从结核杆菌H37Rv株基因组中 ,扩增出分泌蛋白Ag85A成熟蛋白的编码基因 ,用限制性内切酶消化后 ,插入克隆载体 pGEM TEasy中。经酶切鉴定与序列测定证实后 ,以亚克隆法构建于真核表达载体 pcDNA3.1的相应酶切位点。重组质粒肌注免疫小鼠 ,4周后用ELISA法检测抗体滴度。结果　结核分枝杆菌H37Rv株Ag85A成熟蛋白的编码基因经序列测定证实无突变 ;经BamHⅠ和EcoRⅠ双酶切鉴定证实克隆基因正确插入载体 pcDNA3.1。ELISA法检测几何平均滴度为 1∶10 0 0。结论　以Ag85A成熟蛋白的编码基因为基础的DNA疫苗的成功构建和表达 ,为进一步研究其在结核病与肿瘤防治方面的作用奠定了基础     

DNA核酸疫苗诱导小鼠免疫应答的作用

目的　研究DNA核酸疫苗诱导机体免疫应答的能力 ,为揭示其诱导机制奠定基础。方法　采用本室构建的单纯疱疹病毒 2型 (HSV 2 )糖蛋白D的真核表达质粒 pcDNA gD2免疫小鼠 ,通过ELISA法动态检测特异性抗体的产生及小鼠外周血Th1型细胞因子IFN γ、IL 2的水平同时进行病毒攻击实验。结果　pcDNA gD2和HSV 2免疫组小鼠从第 2周开始产生特异性抗糖蛋白D抗体 ,第 4～ 5周达到高峰 ;外周血清中IFN γ、IL 2水平明显高于阴性对照组。病毒攻击实验显示 2周内 pcDNA gD2和HSV 2免疫组小鼠无一只死亡 ,而阴性对照组小鼠全部死亡。结论　pcDNA gD2真核表达质粒可以诱导小鼠产生特异性体液免疫和Th1型细胞免疫 ,并具有免疫保护作用     

葎草花粉主要变应原核酸疫苗pcDNA3.1-LC2的构建及其免疫原性鉴定

目的 构建葎草花粉主要变应原核酸疫苗pcDNA-LC2并鉴定其免疫原性.方法 将pTripIEx2-LC2质粒用EcoRⅠ和HindⅢ双酶切得到LC2葎草花粉变应原基因片段,将其插入pcDNA3.1(-)真核表达载体,在鉴定重组质粒构建成功后,大量制备去内毒素核酸疫苗pcDNA3.1-LC2,应用pcDNA3.1-LC2核酸疫苗免疫小鼠,取免疫血清行双向免疫扩散实验,分析其免疫原性.结果 通过测序确认构建pcDNA3.1-LC2中的672bp的编码框碱基与原序列一致,没有突变及缺失.应用pcDNA3.1-LC2免疫BALB/c小鼠,经双向免疫扩散试验验证pcDNA3.1-LC2在小鼠体内可以表达并产生特异性抗体.结论 成功的构建了葎草花粉主要变应原核酸疫苗pcDNA3.1-LC2,pcDNA3.1- LC2可在小鼠体内表达,并能产生葎草花粉变应原特异性抗体,具有良好的免疫原性.     

HSV-1截短gB基因DNA疫苗的构建及初步鉴定

目的　为有效预防单纯疱疹Ⅰ型病毒 (HSV - 1 )感染 ,给多价DNA疫苗的构建奠定基础 ,构建表达截短HSV - 1 gB糖蛋白的DNA疫苗。方法　用PCR技术从HSV - 1基因组中扩增出编码HSV 1gB糖蛋白 1 - 5 1 7氨基酸序列的一段基因序列 ,通过 pGEM -T中介载体 ,将其插入到真核表达质粒pcDNA 3 1 (+)的CMV启动子下游。结果　构建的重组真核表达质粒可在动物体内正确表达目的基因 ,对HSV - 1病毒攻击具有免疫保护作用。结论　本研究对截短HSV - 1 gB基因DNA疫苗进行了初步的探索性研究 ,也为多价HSV 1DNA疫苗的构建奠定了基础。     

先天性长QT综合征相关人类HERG基因真核表达载体的构建及其功能表达

目的构建先天性长QT综合征相关HERG基因的真核表达载体,观察其在真核细胞中的表达,建立稳定的细胞系,探讨基因的功能。方法原核克隆载体pGEM-HERG经限制性内切酶获得HERG cDNA,将HERG cDNA亚克隆到真核表达载体pcDNA3中,用Lipofectamin2000转染试剂介导将pcDNA3-HERG及荧光真核表达载体pRK5-GFP共转染至HEK-293细胞,利用G-418进行细胞筛选,并用稀释法建立稳定的HEK-HERG细胞系。用全细胞膜片钳技术检测HERG基因的功能表达情况。结果在原核克隆载体pGEM-HERG的基础上构建了HERG的真核表达载体pcDNA3-HERG,并使其在HEK-293细胞中成功表达,建立的HEK-HERG细胞系稳定传代。膜片钳技术检测到了HERG通道电流的表达。结论该方法可成功构建及表达HERG的真核表达载体,为今后突变型HERG的研究奠定了基础。     

Anti-CD3 scFv-B7.1真核表达载体的构建及在COS-7细胞中的初步表达

目的　构建anti CD3scFv B7.1真核表达载体 ,并进行初步表达。方法　采用重叠延伸拼接法 (splicingbyoverlapextention ,SOE)将anti CD3scFv和B7.1(V +C)两个基因片段通过spacer序列连接 ,将融合基因克隆入T载体 ,并测序证实。在此基础上构建真核表达载体pcDNA/anti CD3scFv B7.1,并经脂质体法转染COS 7细胞 ,免疫组织化学法检测表达。结果　获得了序列与预期完全相同的融合基因 ;构建了抗CD3scFv B7.1融合基因真核表达载体 ;在COS 7细胞中获得初步表达。结论　成功构建及表达抗CD3scFv B7.1融合基因真核表达载体 ,为进一步研究抗CD3scFv B7.1双功能分子的生物学活性及用于肿瘤生物治疗奠定了基础     

人TSHR膜外区cDNA克隆及真核表达载体的构建

目的通过生物技术获得促甲状腺激素受体膜外区表达载体,为TSHR膜外区的研究提供有力工具。方法从人甲状腺组织中提取总RNA,RT-PCR分离得到TSHR膜外区cDNA,双酶切后插入真核表达载体pcDNA3.1(+)。结果经酶切鉴定、测序分析表明TSHR膜外区片段与GenBank提供的完全相同。结论成功构建了人TSHR膜外区真核表达载体。     

THE CONSTRUCTION AND PRELIMINARY APPRAISEMENT OF HSV-2 gD GENE DNA VACCINE

Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｔｙｐｅ 2 (ＨＳＶ 2 ) ,ｏｎｅｋｉｎｄｏｆｄｏｕｂｌｅｓｔｒａｎｄｓＤＮＡｖｉｒｕｓ ,ｃａｎｃａｕｓｅｓｅｒｉｏｕｓｗｏｒｌｄｗｉｌｄｉｎｆｅｃｔｉｏｎｓ.Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｇｅｎｏｍ ｉｃＤＮＡｃｏｄｅｓｆｏｒａｐｐｒｏｘｉｍａｔｅｌｙ 80 ｐｒｏｔｅｉｎｓ .Ｍｏｓｔｏｒａｌｌａｒｅｅｘｐｒｅｓｓｅｄｄｕｒｉｎｇｌｙｔｉｃｉｎｆｅｃｔｉｏｎ ,ｍａｎｙａｒｅｆｏｕｎｄｉｎｉｎｔａｃｔｖｉｒｉｏｎｓａｎｄａｔｌｅａｓｔ 11ｈａｖｅｂｅｅｎｉｄｅｎｔ…     

人MCHR1真核表达载体构建及稳定转染HEK293细胞系的建立

目的构建人MCHR1真核表达载体,转染HEK293细胞,建立稳定转染的HEK293细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR1基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1( ),经酶切和测序鉴定后,用脂质体转染法转染HEK293细胞,通过G418筛选,建立稳定转染的HEK293细胞系,用RT-PCR、Western-blot检测MCHR1的表达。结果构建了pcDNA3.1( )/MCHR1真核表达载体,建立了稳定转染的HEK293细胞系,并成功地表达了目的基因。结论真核表达载体成功构建和稳定转染HEK293细胞系的建立为进一步研究MCHR1的功能奠定了良好的实验基础。     

反义HSP70真核表达载体的构建和在人喉癌细胞的表达

目的　进行促凋亡治疗喉癌实验 ,构建和鉴定携带反义HSP70 (heatshockprotein 70 )基因的真核表达载体。方法　将HSP70cDNA反向克隆入 pcDNA 3.1,构建CMV启动子控制的真核表达载体 pcDNA AHSP70 ,用酶切鉴定结果 ;应用该载体转染人喉表皮样癌细胞Hep 2 ,G4 18筛选阳性克隆 ;westernblot和免疫组化检测转染前后瘤细胞的HSP70的表达。检测转染前后瘤细胞的生物学性状。结果　获得 pcDNA AHSP70真核表达载体 ,HSP70反义RNA阻断了Hep 2细胞的HSP70的表达 ,经westernblotting和免疫组化证实 ,实验组瘤细胞不能表达或低表达HSP70 ,而对照和空载体组高表达HSP70。转染反义HSP70的真核表达载体的Hep 2细胞比空载体组和对照组的细胞生长缓慢 ,细胞周期可见实验组出现亚二倍凋亡峰 ,而空载体组没有。结论　本研究成功构建了反义HSP70真核表达载体并在人喉癌细胞得以表达。     

人白介素17受体样分子的克隆及表达

目的　构建带有 6×myc的人白介素 17受体样分子的重组表达质粒 ,以便检测hIL 17RLM L基因在真核细胞中的特异性表达。方法　设计带有酶切位点 (EcoRⅠ和XhoⅠ )的特异性引物 ,用PCR方法扩增hIL 17RLM L片段回收后插入带有 6×myc标签的 pcDNA3.0真核表达载体 ,转染COS7细胞后作Westernblot检测其表达。结果　成功地构建了带有 6×myc标签的 pcDNA3.0 6×myc /hIL 17RLM L重组质粒 ,Westernblot检测到该质粒可在真核细胞中表达。结论　用分子生物学的方法成功地构建了真核表达质粒pcDNA3.0 6×myc /hIL 17RLM L ,使该基因的特异性检测成为可能 ,为进一步研究hIL 17RLM L的生物学功能奠定了基础。     

Eukaryotic expression and biological activity analysis of neuroprotective peptide [Gly14]-Humanin

Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h, then cell morphology, MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h, cell viability decreased to (65.8±5.3)%, and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.     

THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

Objective To construct eukaryutic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humorul and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major rapsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, ETMxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry After BALB/c mice were vaccinated with various recombinant plusmids（pVAX1- L1-E6M3 or pVAX1-L1-E7M3 ） and immunie adjuvants （pLXHDmB7-2 or LTB） through different administration routes （intramuscular or intranasal） , the great cellular immune responses were produced us revealed by delayed-type hypersensitivity （DTH） and lymphocyte proliferation, and the expression of IL-4 and IFN- 7 cells in CD4^＋ and CD8^＋ subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8^＋ IFN-γ^＋ cells number, but CD4^＋ IL4^＋ cell number was higher in intranasul immunization groups. The immunization groups using pLXHDmB7.-2 as adjuvant were superior to other groups in immunorespouse. Conclusion These DNA vaccines produce remarkable cellular and humorul immune responses in the mouse and may provide us prophylatic and therapeutic candidates for HPV induced cancer treatment.     

单纯疱疹病毒1型糖蛋白D核酸疫苗的构建及初步免疫观察

目的　构建用于防治单纯疱疹的核酸疫苗并观察其免疫效果。方法　用PCR反应 ,从HSV 1F株基因组DNA中扩增出 gD蛋白的全部编码序列 ,将其插入 pcDNA 3 1 (+)的PCMV下游 ,构建HSVgD核酸疫苗 ,并用酶切分析和测序鉴定 ;通过中和试验、ELISA方法和攻击试验检测用 pLy D三次肌肉接种小鼠的免疫效果。结果　经鉴定证实了HSV gD核酸疫苗 pLy D的构建 ;实验小鼠产生了对HSV 1特异性的抗体 (抗体滴度 :ELISA法 1∶2 0 3,中和试验法 1∶37) ,并经受了HSV 1病毒的攻击 (存活率 :70 % )。结论　成功构建出了HSV gD核酸疫苗pLy D ,用其接种小鼠可以诱导产生特异性免疫反应 ,并具有良好的保护作用。     

葎草花粉主要变应原核酸疫苗pcDNA3.1-LC2的真核细胞表达和免疫分析

目的 验证葎草花粉主要变应原核酸疫苗(pcDNA3.1-LC2)能否在真核细胞内表达,并进行免疫原性、免疫保护作用、安全性等分析.方法 应用磷酸钙沉淀法将pcDNA3.1-LC2转染至HEK293细胞,提取RNA并行RT-PCR,利用琼脂糖凝胶电泳分离PCR产物,验证所分离出的条带与目的 片段是否一致;应用pcDNA3...     

CONSTRUCTION OF ACTIVE RECOMBINANT CASPASE-3 EUKARYOTIC EXPRESSION PLA SMID AND EFFECT OF r-CASPASE-3 ON APOPTOSIS OF PANCREATIC CARCINOMA CELLS

Ａｓｅｒｉｅｓｏｆｃｙｓｔｅｉｎｅｐｒｏｔｅａｓｅ(ｃａｓｐａｓｅ)ｔｈａｔｐｌａｙｉｍｐｏｒｔａｎｔｒｏｌｅｓｄｕｒｉｎｇｔｈｅｐｒｏｃｅｓｓｏｆｍａｍｍａｌｉａｎｃｅｌｌａｐｏｐｔｏｓｉｓｈａｖｅｂｅｅｎｃｌｏｎｅｄ .Ｃａｓｐａｓｅ 3(ＣＰＰ32 /Ｙａ ｍａ/ａｐｏｐａｉｎ)ｈａｓｂｅｅｎｔｈｏｕｇｈｔｔｏｂｅｔｈｅｋｅｙｐｒｏｔｅａｓｅａｍｏｎｇｃａｓｐａｓｅｆａｍｉｌｙ .Ａｃｔｉｖｅｃａｓｐａｓｅ 3ｃａｎａｆｆｅｃｔｔｈｅｏｔｈｅｒｍｅｍｂｅｒｓｏｆｃａｓｐａｓｅｓｆａｍｉｌｙ ,ｍｏｒｅｏｖｅｒ…     

Construction and characterization of hGM-CSF-expressing K-562 cell line

Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The eukaryocyte expressing plasmid pcDNA3. 1/GM-CSF was first constructed and its accuracy was verified through sequencing. The pcDNA3. 1/GM-CSF was transfected into COS-7 cells to verify GM-CSF expression and cytokine activity using TF-1 cell line. Then the plasmid was transfected into K-562 cell line using liposome method, and was selected under G-418 and sub-cloned by limiting dilution. GM-CSF product from the monoclone GM-CSF-K-562 cell lines was quantified using ELISA method. Results We successfully constructed the hGM-CSF eukaryocyte expressing plasmid and hGM-CSF expressing K-562 cell line. Conclusion The construction of K-562/GM-CSF line will simplify the preparation of tumor vaccine, thus facilitating the application of tumor vaccination therapy in clinical application.     

HPV-6/11 L1/E6、HPV-6/11 L1/E7嵌合DNA疫苗质粒的构建

目的　构建HPV 6 / 11L1/E6、HPV 6 / 11L1/E7预防、治疗性DNA疫苗质粒。方法　运用PCR扩增HPV 6 /11L1、E6、E7序列 ,PCR产物连接至pGEMT Easy质粒 ,测序鉴定正确后 ,分别连接至真核表达载体pVAX ,再用酶切、连接而构建成HPV 6 / 11L1 E6 /E7 pVAX质粒。运用电穿孔法将所获得质粒转染COS 7细胞 ,免疫组化检测蛋白表达情况。结果　所构建质粒的插入目的片段测序正确 ;免疫组化检测在胞浆胞核可见棕黄色点状阳性产物沉积。结论　所构建的HPV 6 / 11L1 E6 /E7 pVAX融合蛋白表达质粒可在体外表达L1 E6 /L1 E7蛋白 ,为今后进行DNA疫苗的动物实验及临床实验研究做好准备     

外源性p16基因对肾癌细胞周期及体外增殖的影响

目的　观察外源性 p16基因对肾癌细胞周期的影响及其对肾癌细胞的生长抑制作用。方法　利用携带人 p16基因的真核表达载体,采用脂质体介导法将其导入人肾癌细胞系 GRC- 1 中,观察其对瘤细胞的作用。结果　①免疫组织化学方法检测显示转染 p16基因的GRC 1细胞可显著表达 p16蛋白。②流式细胞仪检测证明,外源性 p16 基因可在肾癌细胞周期G1期到S期发挥抑制作用。③与对照组相比,转染 p16 基因的 GRC- 1 细胞生长明显受到抑制。结论　外源性 p16基因具有细胞周期特异性地抑制肾癌细胞生长的作用。     

重组小鼠血管抑素基因在胆囊癌细胞中的表达及对血管内皮细胞增殖的抑制作用

目的　研究重组小鼠血管抑素基因真核表达质粒转染的胆囊癌细胞能否表达具有抑制血管内皮细胞生长活性的血管抑素蛋白。方法　应用脂质体LipofectAMINE 2 0 0 0将重组小鼠血管抑素基因的真核表达质粒 pcDNA3.1(+) angiostatin转染胆囊癌细胞株GBC SD ,G4 18抗性筛选 ;以Westernblot检测血管抑素的表达 ,以血管内皮细胞增殖分析检测其活性。结果　经Westernblot检测 ,得到高表达血管抑素的胆囊癌细胞克隆 ,其培养上清能有效抑制bFGF刺激的血管内皮细胞增殖 (P <0 .0 5 )。结论　小鼠血管抑素基因真核表达质粒 pcDNA3.1(+) angiostatin转导的胆囊癌细胞能够表达具有生物学活性的血管抑素蛋白 ,在胆囊癌的血管抑制基因治疗中有潜在临床应用价值