汉坦病毒嵌合基因G1S0.7、G2S0.7重组腺病毒的改建、表达及其免疫学特性的比较研究
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摘要
肾综合征出血热(Hemorrhagic fever with renal syndrome, HFRS)是由汉坦病毒(Hantavirus, HTV)引起的急性病毒性传染病,临床上尚缺乏特异有效的治疗药物,因此其防治任务十分艰巨。现有的HFRS疫苗主要是鼠脑来源和细胞来源的灭活疫苗,虽然该疫苗在HFRS防治方面起到了积极作用,但仍存在明显不足,主要是其诱导机体产生中和抗体的能力较弱,中和抗体滴度不高,细胞免疫水平亦不高。因此需要更为有效的新型疫苗来弥补现有疫苗的不足。
HTV的囊膜糖蛋白(Glycoprotein, GP)是HFRS基因工程疫苗基础研究中的重要组分。研究表明,GP上有中和抗原表位,能诱导机体产生中和抗体从而起到保护作用。但是GP免疫原性较弱,诱生的抗体出现晚,滴度低;而HTV的核衣壳蛋白(Nucleocapsid protein, NP)被认为是其三个结构蛋白中免疫原性强的蛋白,且NP诱生的抗体出现早,滴度高。虽然现有研究对其是否具有中和抗原表位存在争议,但是大量研究证实NP对HTV感染的实验动物具有保护作用。此外NP上还存在多个针对HTV的CTL表位,因此NP在机体的细胞免疫应答中发挥重要作用。我室以往研究表明,NP的抗原结合位点主要位于S基因0.7 kb片段(即编码NP氨基端1~274aa片段)上。将该截短片段克隆进原核载体并用表达产物免疫小鼠,动物实验结果显示与完整NP免疫效果基本相同;进一步构建了M片段与S0.7的嵌合基因(G1S0.7、G2S0.7),并在不同系统中表达,同时对其免疫学特性进行检测,结果显示嵌合基因/融合蛋白能有效的诱导机体体液和细胞免疫应答,且其在腺病毒载体中的免疫效果优于其他系统。然而研究中我们发现各个表达系统存在融合蛋白表达水平不高、表达产物免疫小鼠后细胞免疫水平也相对较低的问题。因此有必要采取有效策略来解决这一问题。
本课题在我室前期工作基础上,以人5型复制缺陷型腺病毒Adeno-XTM系统为基础,利用基因重组技术,对含HTV嵌合基因G1S0.7、G2S0.7的pShuttle转移载体进行改建和修饰,包括将原有人巨细胞病毒早期增强子/启动子(Human cytomegalovirus early enhancer/promoter, CMV)替换为CMV与鸡β肌动蛋白的杂合启动子(hybrid of Human cytomegalovirus early enhancer and chickenβ-actin promoter, CAG),单独插入土拨鼠肝炎病毒转录后调控元件(Woodchuck Hepatitis Virus post-transcriptional regulatory element, WPRE),或替换启动子的同时插入WPRE,以期在转录及转录后调控阶段提高抗原的表达水平;构建病毒载体并包装重组腺病毒,鉴定并比较不同重组腺病毒中融合蛋白G1S0.7、G2S0.7的表达及其水平的高低;大量制备、纯化各重组腺病毒,免疫小鼠,并对其免疫学特性进行系统研究。
1、设计并合成CAG启动子,替换pShuttle转移载体CMV启动子,获得含HTV嵌合基因G1S0.7、G2S0.7的改建重组腺病毒转移载体,分别命名为G1S0.7-pCAG和G2S0.7-pCAG;设计并合成WPRE,插入重组pShuttle载体中目的片段3端,获得含HTV嵌合基因G1S0.7、G2S0.7的改建重组腺病毒转移载体,分别命名为G1S0.7-WPRE和G2S0.7-WPRE;同时替换启动子并插入WPRE,获得含HTV嵌合基因G1S0.7、G2S0.7的改建重组腺病毒转移载体,分别命名为G1S0.7-pCAG-WPRE和G2S0.7-pCAG-WPRE。
2、将上述改建的重组转移载体分别克隆入腺病毒载体骨架pAdeno-X DNA,并经XhoⅠ、PCR及PI-SceⅠ和I-CeuⅠ双酶切鉴定,获得阳性重组腺病毒DNA。转染HEK 293细胞,包装重组腺病毒,分别命名rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG、rAd-G1S0.7-WPRE、rAd-G2S0.7-WPRE、rAd-G1S0.7-pCAG-WPRE和rAd-G2S0.7-pCAG-WPRE。
3、扩增、纯化并测定重组腺病毒滴度,用IFA和Western blot对表达产物进行鉴定,同时加入未改建的含嵌合基因重组腺病毒rAd-G1S0.7、rAd-G2S0.7作为对照。结果显示,所有重组腺病毒感染HEK 293细胞后,用特异性抗体均可检测到胞浆内的绿色荧光,表明所有重组腺病毒均可表达融合蛋白G1S0.7和G2S0.7,其中替换启动子的rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG感染细胞荧光亮度最强;Western blot结果证实,替换了CAG启动子的rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG表达融合蛋白的水平最高,分别是其他改建及未改建的含嵌合基因G1S0.7、G2S0.7重组腺病毒的2.1倍和2.3倍。
4、将各组重组腺病毒经腹腔注射免疫C57BL/6小鼠,并同时设立HFRS灭活疫苗、Adenovirus-Lac Z和正常小鼠对照组。通过ELISA、微量细胞培养中和试验、ELISPOT及CTL杀伤实验观察重组腺病毒的免疫学特性。ELISA检测结果显示,免疫小鼠血清可以检测到最高效价分别为1: 80和1: 320的特异性抗汉滩病毒( Hantaan virus, HTNV ) GP、NP抗体,rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG分别高于其他改建和未改建的含嵌合基因G1S0.7、G2S0.7的重组腺病毒;微量细胞培养中和试验结果显示,各实验组小鼠血清中均可检测到不同滴度的中和抗体(1: 10~1: 40),其中rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG均高于疫苗对照组(1: 10~1: 20);ELISPOT检测结果显示,各实验组小鼠脾细胞分泌细胞因子水平不同,其中rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG分泌IFN-γ和IL-2水平分别高于其他改建及未改建含嵌合基因G1S0.7、G2S0.7的重组腺病毒及疫苗对照组(P<0.05),TNF-α和IL-10差异不明显;CTL杀伤实验提示,各实验组均可诱导CTL杀伤作用,且随着效靶比的升高,杀伤效应也相应升高,其中在效靶比为100: 1和50: 1时, rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG诱导的杀伤活性分别高于其他改建及未改建含嵌合基因G1S0.7、G2S0.7的重组腺病毒及疫苗对照组(P<0.05)。
综上所述,通过替换转移载体启动子,我们获得融合蛋白表达水平高于其他组的重组腺病毒rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG;动物实验结果显示,这两各重组腺病毒诱导体液免疫应答和细胞免疫应答能力高于其他实验组,特别是细胞免疫应答水平,亦高于本研究中灭活疫苗对照组。下一步,我们将进一步改建重组腺病毒转移,通过加入泛素或者HSP70等免疫佐剂,以期获得能诱导更高细胞免疫应答的重组腺病毒。
Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantaviruses (HTV), and there is no specific treatment right now. There are a few inactivated vaccines licensed for use in China, but their prophylactic effect has prompted mixed reviews. One clear problem is the vaccines’poor immunogenicity for eliciting neutralizing antibodies and cell-mediated immunity. It is clearly indicates a need for developing more effective vaccines.
It is well demonstrated that GP is a main candidate protein for the HTV genetically engineered vaccine: It plays an important role in stimulating neutralizing antibodies and in protecting humans and animals from HTV infection; however, its immunogenicity is weak, the antibody elicited by GP is produced later, and the titer is low. Among the structural proteins, NP has the strongest immunogenicity and it can elicit a high titer and a long-lasting antibody response. It is disputed that NP contains neutralizing epitopes, but the fact that NP can protect animals from HTV infection is totally confirmed. Besides, NP contains antigenic sites associated with cytotoxic T-lymphocyte (CTL) responses. Thererfore, NP plays an important role in evoking cellular immune response against HTV infection. Previous work has demonstrated that the antigenic sites of NP are mainly distributed at the 0.7 kb fragment of the S segment, which is close to the N-terminus. Mice immunized with the truncated protein expressed in prokaryotic vector could produce the same immunological effect as immunized with the complete NP. Further experiments indicated that mice immunized with the fusion proteins G1S0.7 (G1 of the M segment and a 0.7 kb fragment of the S segment) and G2S0.7 (G2 of the M segment and a 0.7 kb fragment of the S segment) effectively elicit specific anti-NP, anti-GP and neutralizing antibodies and a cellular immune response. And the immune response produced by the recombinant adenovirus containing the chemiric genes was more efficient than other systems. But the expreesion level of the fused proteins was not satisfied, and the cell-mediated immunity of mice which were immuned with the expressed products was still low. Efficient strategies are need for the problems.
In this study, we chose the human adenoviral type 5 replication-incompetent systems to express the fusion proteins G1S0.7 and G2S0.7. To improve the expression levels, we made several modifications to the adenoviral vector pShuttle, including replacing the CMV promoter (Human cytomegalovirus early enhancer/promoter) with CAG promoter (hybrid of Human cytomegalovirus early enhancer and chickenβ-actin promoter). We also incorporated WPRE (Woodchuck Hepatitis Virus post-transcriptional regulatory element) alone, or in conjunction with, the new promoter CAG. After packaging the recombinant adenoviruses and infecting HEK 293 cells, we identified the fusion proteins and compared the expression level as well as immunized mice with recombinant adenoviruses, a series of immunological assays were taken and the immunological characteristics were studied.
1. By designing and synthesizing the CAG promoter, the classic CMV promoter of the adenoviral vectors pShuttle containing chimeric genes G1S0.7 or G2S0.7 were replaced, naming G1S0.7-pCAG and G2S0.7-pCAG; Designed and synthesized the WPRE, incorporated at the 3′UTR of pShuttle carrying chimeric genes G1S0.7 or G2S0.7, naming G1S0.7-WPRE and G2S0.7-WPRE; By both replacing the promoter and incorporating WPRE, the reconstructed vectors containing chimeric genes G1S0.7 or G2S0.7 were modified, naming G1S0.7-pCAG-WPRE and G2S0.7-pCAG-WPRE, respectively.
2. Reconstructed pShuttle vestors containing chimeric genes, and the pAdeno-X DNA were all digested with PI-SceⅠand I-CeuⅠ, and the ligation products were transformated into E.coli JM109. Clones were identified by XhoⅠdigestion, PCR amplified and PI-SceⅠand I-CeuⅠdouble digestion. After transfected HEK 293 cells, the recombinant Adenoviruses were packaged, naming rAd-G1S0.7-pCAG, rAd-G2S0.7-pCAG, rAd-G1S0.7-WPRE, rAd-G2S0.7-WPRE, rAd-G1S0.7-pCAG-WPRE and rAd-G2S0.7-pCAG-WPRE.
3. By purifing and determinating the viral titers, immunofluorescence assays and Western blot were employed to identify the expression products, the unmodified recombinant adenoviruses rAd-G1S0.7 and rAd-G2S0.7 were used as controls. A positive fluorescence could be observed in 293 cells 48 hrs after infection in IFA. The most intense fluorescence for the target proteins were all observed in vector with the CAG promoter groups. Western blot results indicated that the reconstructed vectors with a CAG promoter and carrying the chemiric genes G1S0.7 or G2S0.7, expressed 2.1-fold and 2.3-fold more protein than the unmodified vectors, respectively.
4. C57BL/6 mice were inoculated by intraperitoneal injection, HFRS inactivated vaccine, Adenovirus-Lac Z and normal mice were employed as controls. ELISA, microcell-cultured neutralization test, ELISPOT and CTL assay were performed for the immunological characteristics of recombinant adenoviruses. The results showed that among all the experimental groups the titers of mice immunized with rAd-G1S0.7-pCAG were the highest, which were 1: 80 and 1: 320, respectively. In the microcell-cultured neutralization test, a low titer of neutralization antibodies were observed in all experimental groups (1: 10~1: 40), of which the rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were higher than the vaccine control (1: 10~1: 20). The levels of cytokines stimulated by splenocytes were quite different: the INF-γand IL-2 of rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were higher than the other experimental groups and vaccine control (P<0.05), while TNF-αand IL-10 did not change remarkedly during all the groups. Cell-mediated cytotoxicity assay results indicated that all the recombinant adenoviruses induced specific cytotoxic effects on target cells, which was enhanced in accord with the rise of E/T ration. Among all the experimental groups the specific cytotoxic effects of the rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were stronger than the other experimental groups and vaccine control at the E/T ration of 100: 1 and 50: 1(P<0.05).
By replacing the promoter, we obtained 2 recombinant adenviruese rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG, which could express the fusion proteins in a relative high level. Animal experiments showed that these recombinant adenviruses induced a stronger humoral immunoresponse and cellullar immunologic response, especially the cellullar immunologic response, which were stronger than the vaccine control. Next step, we hope to obtain more recombinant adenoviruses which could induce effective cell-mediated immunity by inserting the adjuvant such as ubiquitin and HSP70.
HTV的囊膜糖蛋白(Glycoprotein, GP)是HFRS基因工程疫苗基础研究中的重要组分。研究表明,GP上有中和抗原表位,能诱导机体产生中和抗体从而起到保护作用。但是GP免疫原性较弱,诱生的抗体出现晚,滴度低;而HTV的核衣壳蛋白(Nucleocapsid protein, NP)被认为是其三个结构蛋白中免疫原性强的蛋白,且NP诱生的抗体出现早,滴度高。虽然现有研究对其是否具有中和抗原表位存在争议,但是大量研究证实NP对HTV感染的实验动物具有保护作用。此外NP上还存在多个针对HTV的CTL表位,因此NP在机体的细胞免疫应答中发挥重要作用。我室以往研究表明,NP的抗原结合位点主要位于S基因0.7 kb片段(即编码NP氨基端1~274aa片段)上。将该截短片段克隆进原核载体并用表达产物免疫小鼠,动物实验结果显示与完整NP免疫效果基本相同;进一步构建了M片段与S0.7的嵌合基因(G1S0.7、G2S0.7),并在不同系统中表达,同时对其免疫学特性进行检测,结果显示嵌合基因/融合蛋白能有效的诱导机体体液和细胞免疫应答,且其在腺病毒载体中的免疫效果优于其他系统。然而研究中我们发现各个表达系统存在融合蛋白表达水平不高、表达产物免疫小鼠后细胞免疫水平也相对较低的问题。因此有必要采取有效策略来解决这一问题。
本课题在我室前期工作基础上,以人5型复制缺陷型腺病毒Adeno-XTM系统为基础,利用基因重组技术,对含HTV嵌合基因G1S0.7、G2S0.7的pShuttle转移载体进行改建和修饰,包括将原有人巨细胞病毒早期增强子/启动子(Human cytomegalovirus early enhancer/promoter, CMV)替换为CMV与鸡β肌动蛋白的杂合启动子(hybrid of Human cytomegalovirus early enhancer and chickenβ-actin promoter, CAG),单独插入土拨鼠肝炎病毒转录后调控元件(Woodchuck Hepatitis Virus post-transcriptional regulatory element, WPRE),或替换启动子的同时插入WPRE,以期在转录及转录后调控阶段提高抗原的表达水平;构建病毒载体并包装重组腺病毒,鉴定并比较不同重组腺病毒中融合蛋白G1S0.7、G2S0.7的表达及其水平的高低;大量制备、纯化各重组腺病毒,免疫小鼠,并对其免疫学特性进行系统研究。
1、设计并合成CAG启动子,替换pShuttle转移载体CMV启动子,获得含HTV嵌合基因G1S0.7、G2S0.7的改建重组腺病毒转移载体,分别命名为G1S0.7-pCAG和G2S0.7-pCAG;设计并合成WPRE,插入重组pShuttle载体中目的片段3端,获得含HTV嵌合基因G1S0.7、G2S0.7的改建重组腺病毒转移载体,分别命名为G1S0.7-WPRE和G2S0.7-WPRE;同时替换启动子并插入WPRE,获得含HTV嵌合基因G1S0.7、G2S0.7的改建重组腺病毒转移载体,分别命名为G1S0.7-pCAG-WPRE和G2S0.7-pCAG-WPRE。
2、将上述改建的重组转移载体分别克隆入腺病毒载体骨架pAdeno-X DNA,并经XhoⅠ、PCR及PI-SceⅠ和I-CeuⅠ双酶切鉴定,获得阳性重组腺病毒DNA。转染HEK 293细胞,包装重组腺病毒,分别命名rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG、rAd-G1S0.7-WPRE、rAd-G2S0.7-WPRE、rAd-G1S0.7-pCAG-WPRE和rAd-G2S0.7-pCAG-WPRE。
3、扩增、纯化并测定重组腺病毒滴度,用IFA和Western blot对表达产物进行鉴定,同时加入未改建的含嵌合基因重组腺病毒rAd-G1S0.7、rAd-G2S0.7作为对照。结果显示,所有重组腺病毒感染HEK 293细胞后,用特异性抗体均可检测到胞浆内的绿色荧光,表明所有重组腺病毒均可表达融合蛋白G1S0.7和G2S0.7,其中替换启动子的rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG感染细胞荧光亮度最强;Western blot结果证实,替换了CAG启动子的rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG表达融合蛋白的水平最高,分别是其他改建及未改建的含嵌合基因G1S0.7、G2S0.7重组腺病毒的2.1倍和2.3倍。
4、将各组重组腺病毒经腹腔注射免疫C57BL/6小鼠,并同时设立HFRS灭活疫苗、Adenovirus-Lac Z和正常小鼠对照组。通过ELISA、微量细胞培养中和试验、ELISPOT及CTL杀伤实验观察重组腺病毒的免疫学特性。ELISA检测结果显示,免疫小鼠血清可以检测到最高效价分别为1: 80和1: 320的特异性抗汉滩病毒( Hantaan virus, HTNV ) GP、NP抗体,rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG分别高于其他改建和未改建的含嵌合基因G1S0.7、G2S0.7的重组腺病毒;微量细胞培养中和试验结果显示,各实验组小鼠血清中均可检测到不同滴度的中和抗体(1: 10~1: 40),其中rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG均高于疫苗对照组(1: 10~1: 20);ELISPOT检测结果显示,各实验组小鼠脾细胞分泌细胞因子水平不同,其中rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG分泌IFN-γ和IL-2水平分别高于其他改建及未改建含嵌合基因G1S0.7、G2S0.7的重组腺病毒及疫苗对照组(P<0.05),TNF-α和IL-10差异不明显;CTL杀伤实验提示,各实验组均可诱导CTL杀伤作用,且随着效靶比的升高,杀伤效应也相应升高,其中在效靶比为100: 1和50: 1时, rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG诱导的杀伤活性分别高于其他改建及未改建含嵌合基因G1S0.7、G2S0.7的重组腺病毒及疫苗对照组(P<0.05)。
综上所述,通过替换转移载体启动子,我们获得融合蛋白表达水平高于其他组的重组腺病毒rAd-G1S0.7-pCAG和rAd-G2S0.7-pCAG;动物实验结果显示,这两各重组腺病毒诱导体液免疫应答和细胞免疫应答能力高于其他实验组,特别是细胞免疫应答水平,亦高于本研究中灭活疫苗对照组。下一步,我们将进一步改建重组腺病毒转移,通过加入泛素或者HSP70等免疫佐剂,以期获得能诱导更高细胞免疫应答的重组腺病毒。
Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantaviruses (HTV), and there is no specific treatment right now. There are a few inactivated vaccines licensed for use in China, but their prophylactic effect has prompted mixed reviews. One clear problem is the vaccines’poor immunogenicity for eliciting neutralizing antibodies and cell-mediated immunity. It is clearly indicates a need for developing more effective vaccines.
It is well demonstrated that GP is a main candidate protein for the HTV genetically engineered vaccine: It plays an important role in stimulating neutralizing antibodies and in protecting humans and animals from HTV infection; however, its immunogenicity is weak, the antibody elicited by GP is produced later, and the titer is low. Among the structural proteins, NP has the strongest immunogenicity and it can elicit a high titer and a long-lasting antibody response. It is disputed that NP contains neutralizing epitopes, but the fact that NP can protect animals from HTV infection is totally confirmed. Besides, NP contains antigenic sites associated with cytotoxic T-lymphocyte (CTL) responses. Thererfore, NP plays an important role in evoking cellular immune response against HTV infection. Previous work has demonstrated that the antigenic sites of NP are mainly distributed at the 0.7 kb fragment of the S segment, which is close to the N-terminus. Mice immunized with the truncated protein expressed in prokaryotic vector could produce the same immunological effect as immunized with the complete NP. Further experiments indicated that mice immunized with the fusion proteins G1S0.7 (G1 of the M segment and a 0.7 kb fragment of the S segment) and G2S0.7 (G2 of the M segment and a 0.7 kb fragment of the S segment) effectively elicit specific anti-NP, anti-GP and neutralizing antibodies and a cellular immune response. And the immune response produced by the recombinant adenovirus containing the chemiric genes was more efficient than other systems. But the expreesion level of the fused proteins was not satisfied, and the cell-mediated immunity of mice which were immuned with the expressed products was still low. Efficient strategies are need for the problems.
In this study, we chose the human adenoviral type 5 replication-incompetent systems to express the fusion proteins G1S0.7 and G2S0.7. To improve the expression levels, we made several modifications to the adenoviral vector pShuttle, including replacing the CMV promoter (Human cytomegalovirus early enhancer/promoter) with CAG promoter (hybrid of Human cytomegalovirus early enhancer and chickenβ-actin promoter). We also incorporated WPRE (Woodchuck Hepatitis Virus post-transcriptional regulatory element) alone, or in conjunction with, the new promoter CAG. After packaging the recombinant adenoviruses and infecting HEK 293 cells, we identified the fusion proteins and compared the expression level as well as immunized mice with recombinant adenoviruses, a series of immunological assays were taken and the immunological characteristics were studied.
1. By designing and synthesizing the CAG promoter, the classic CMV promoter of the adenoviral vectors pShuttle containing chimeric genes G1S0.7 or G2S0.7 were replaced, naming G1S0.7-pCAG and G2S0.7-pCAG; Designed and synthesized the WPRE, incorporated at the 3′UTR of pShuttle carrying chimeric genes G1S0.7 or G2S0.7, naming G1S0.7-WPRE and G2S0.7-WPRE; By both replacing the promoter and incorporating WPRE, the reconstructed vectors containing chimeric genes G1S0.7 or G2S0.7 were modified, naming G1S0.7-pCAG-WPRE and G2S0.7-pCAG-WPRE, respectively.
2. Reconstructed pShuttle vestors containing chimeric genes, and the pAdeno-X DNA were all digested with PI-SceⅠand I-CeuⅠ, and the ligation products were transformated into E.coli JM109. Clones were identified by XhoⅠdigestion, PCR amplified and PI-SceⅠand I-CeuⅠdouble digestion. After transfected HEK 293 cells, the recombinant Adenoviruses were packaged, naming rAd-G1S0.7-pCAG, rAd-G2S0.7-pCAG, rAd-G1S0.7-WPRE, rAd-G2S0.7-WPRE, rAd-G1S0.7-pCAG-WPRE and rAd-G2S0.7-pCAG-WPRE.
3. By purifing and determinating the viral titers, immunofluorescence assays and Western blot were employed to identify the expression products, the unmodified recombinant adenoviruses rAd-G1S0.7 and rAd-G2S0.7 were used as controls. A positive fluorescence could be observed in 293 cells 48 hrs after infection in IFA. The most intense fluorescence for the target proteins were all observed in vector with the CAG promoter groups. Western blot results indicated that the reconstructed vectors with a CAG promoter and carrying the chemiric genes G1S0.7 or G2S0.7, expressed 2.1-fold and 2.3-fold more protein than the unmodified vectors, respectively.
4. C57BL/6 mice were inoculated by intraperitoneal injection, HFRS inactivated vaccine, Adenovirus-Lac Z and normal mice were employed as controls. ELISA, microcell-cultured neutralization test, ELISPOT and CTL assay were performed for the immunological characteristics of recombinant adenoviruses. The results showed that among all the experimental groups the titers of mice immunized with rAd-G1S0.7-pCAG were the highest, which were 1: 80 and 1: 320, respectively. In the microcell-cultured neutralization test, a low titer of neutralization antibodies were observed in all experimental groups (1: 10~1: 40), of which the rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were higher than the vaccine control (1: 10~1: 20). The levels of cytokines stimulated by splenocytes were quite different: the INF-γand IL-2 of rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were higher than the other experimental groups and vaccine control (P<0.05), while TNF-αand IL-10 did not change remarkedly during all the groups. Cell-mediated cytotoxicity assay results indicated that all the recombinant adenoviruses induced specific cytotoxic effects on target cells, which was enhanced in accord with the rise of E/T ration. Among all the experimental groups the specific cytotoxic effects of the rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were stronger than the other experimental groups and vaccine control at the E/T ration of 100: 1 and 50: 1(P<0.05).
By replacing the promoter, we obtained 2 recombinant adenviruese rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG, which could express the fusion proteins in a relative high level. Animal experiments showed that these recombinant adenviruses induced a stronger humoral immunoresponse and cellullar immunologic response, especially the cellullar immunologic response, which were stronger than the vaccine control. Next step, we hope to obtain more recombinant adenoviruses which could induce effective cell-mediated immunity by inserting the adjuvant such as ubiquitin and HSP70.
引文
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