Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed ...Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.展开更多
In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its pa- rental virulent strain (EIAV LN) were inserted respe...In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its pa- rental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost proce- dures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag pro- teins. The results showed that the specific lysis of CTL re- sponses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses in- duced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to pro- duce powerful cellular responses. These results lay an im- portant foundation for the development of a new EIAV ge- netic engineering vaccine.展开更多
A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will b...A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.展开更多
基于DNA载体的RNA i技术可以较长时间高效地抑制基因的活性。利用家蚕核型多角体病毒ie-1基因启动子,构建了在细胞内能转录形成与ie-1基因+19^+446区域互补同源的发夹状dsRNA的RNA i DNA载体。研究结果表明,该载体DNA在体外、体内均能...基于DNA载体的RNA i技术可以较长时间高效地抑制基因的活性。利用家蚕核型多角体病毒ie-1基因启动子,构建了在细胞内能转录形成与ie-1基因+19^+446区域互补同源的发夹状dsRNA的RNA i DNA载体。研究结果表明,该载体DNA在体外、体内均能较好地抑制家蚕核型多角体病毒在细胞中的复制。展开更多
目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电...目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电泳法分析质粒 DNA 的超螺旋构象比例和残留 RNA 含量;采用阴离子交换色谱法分析 HPLC 纯度;采用 ELISA 法测定hPK～5蛋白的表达量;采用内皮细胞迁移法测定 hPK-5蛋白的生物学活性。结果:重组质粒 DNA 的限制性酶切片段大小与理论值一致;插入基因 PCR 扩增片段大小与理论值相符;质粒 DNA 含量为1.12 mg·mL^(-1);A_(260)/A_(280)比值为1.83;琼脂糖凝胶电泳法测定超螺旋构象质粒的比例为95.4%;HPLC 测定超螺旋质粒 DNA 占93.8%,开环质粒 DNA 占6.2%,琼脂糖凝胶电泳法测定木检出 RNA 残留;没有检出其他杂质;以2μg重组 hPK-5质粒转染5×10~5Hela 细胞48 h 后,2 mL 培养上清中 hPK-5蛋白浓度为109 ng·mL^(-1);以培养上清进行内皮细胞迁移试验,转染重组 hPK-5质粒 DNA 组迁移的内皮细胞数明显少于对照绀(P<0.05)。其他项目均符合相应的规定。结论:建立了重组 hPK-5质粒 DNA 基因治疗制剂的检定方法及其质量标准,为有效控制该类产品的质量打下基础。展开更多
文摘Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.
文摘In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its pa- rental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost proce- dures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag pro- teins. The results showed that the specific lysis of CTL re- sponses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses in- duced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to pro- duce powerful cellular responses. These results lay an im- portant foundation for the development of a new EIAV ge- netic engineering vaccine.
文摘A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.
文摘基于DNA载体的RNA i技术可以较长时间高效地抑制基因的活性。利用家蚕核型多角体病毒ie-1基因启动子,构建了在细胞内能转录形成与ie-1基因+19^+446区域互补同源的发夹状dsRNA的RNA i DNA载体。研究结果表明,该载体DNA在体外、体内均能较好地抑制家蚕核型多角体病毒在细胞中的复制。
文摘目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电泳法分析质粒 DNA 的超螺旋构象比例和残留 RNA 含量;采用阴离子交换色谱法分析 HPLC 纯度;采用 ELISA 法测定hPK～5蛋白的表达量;采用内皮细胞迁移法测定 hPK-5蛋白的生物学活性。结果:重组质粒 DNA 的限制性酶切片段大小与理论值一致;插入基因 PCR 扩增片段大小与理论值相符;质粒 DNA 含量为1.12 mg·mL^(-1);A_(260)/A_(280)比值为1.83;琼脂糖凝胶电泳法测定超螺旋构象质粒的比例为95.4%;HPLC 测定超螺旋质粒 DNA 占93.8%,开环质粒 DNA 占6.2%,琼脂糖凝胶电泳法测定木检出 RNA 残留;没有检出其他杂质;以2μg重组 hPK-5质粒转染5×10~5Hela 细胞48 h 后,2 mL 培养上清中 hPK-5蛋白浓度为109 ng·mL^(-1);以培养上清进行内皮细胞迁移试验,转染重组 hPK-5质粒 DNA 组迁移的内皮细胞数明显少于对照绀(P<0.05)。其他项目均符合相应的规定。结论:建立了重组 hPK-5质粒 DNA 基因治疗制剂的检定方法及其质量标准,为有效控制该类产品的质量打下基础。
