表达H9亚型禽流感病毒HA基因的重组禽痘病毒的构建及其免疫效力的评估
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摘要
禽流感(Avian Influenza,AI)是由A型流感病毒引起的禽类的感染和/或疾病综合征,分为高致病性禽流感(HPAI)和低致病性禽流感(LPAI)。H9亚型低致病性禽流感近年来在我国部分地区广泛流行,给养禽业造成了严重的经济损失。重组病毒载体疫苗是防制禽流感的疫苗之一,许多株表达各种亚型禽流感病毒HA基因的重组禽痘病毒疫苗免疫后均可对相应亚型的不同毒株(包括高、低致病力毒株)的攻击产生近100%的免疫保护性,而且重组病毒载体疫苗应用后不会干扰以传统检测方法为基础的禽流感疫情监测,具有较好的应用前景。
本实验构建的禽痘病毒通用转移载体pSY681- gfp-gpt含有黄嘌呤鸟嘌呤磷酸转移酶(gfp)和绿色荧光蛋白(gpt)两个报告基因及背对的禽痘病毒晚期启动子P11和早期启动子P7.5,P11启动gfp和gpt两个基因,P7.5用于启动外源基因,在早期启动子P7.5下游引入NotI和Afl II两个酶切位点,用于外源基因的插入。将H9亚型禽流感病毒的HA基因插入到此载体,从而构建出表达HA基因的重组禽痘病毒转移载体pSY681- gfp-gpt-HA。应用TransIT?-LT1 Transfection Reagent将此转移载体转染已感染禽痘病毒S-FPV-017的鸡胚成纤维细胞,利用gfp和gpt同时进行双重筛选、数轮蚀斑纯化后获得了重组病毒rFPV -HA9。通过PCR、Western-blot鉴定,结果证明,rFPV -HA9能稳定表达AIV HA蛋白。用该重组病毒进行鸡翅刺种免疫21日龄SPF鸡,免疫3周后,每只鸡用106EID50的H9亚型LPAIV A/Chicken/Shanghai/10/01(H9N2)攻毒,分别于攻毒后第3、5、7天采集喉头及泄殖腔拭子进行病毒分离、滴定检测排毒情况,同时检测免疫后、攻毒前及攻毒后血清HI抗体、AGP抗体的动态变化,对重组病毒的免疫保护效果进行全面评估。结果,免疫组在免疫后一周就检测到了HI抗体,免疫后三周HI抗体水平达到了7log2以上,而且其AGP抗体均为阴性;攻毒后,免疫组在第三天只有15 %的鸡只在喉头有低水平的一过性的排毒,第五天全部停止排毒,其AGP抗体均为阴性。而对照组鸡只攻毒后第三天和第五天100%排毒,其AGP抗体均为阳性。通过Hl抗体水平和抑制排毒效力反应了本实验构建的重组禽痘病毒能够产生很好的免疫保护力。
总之,通过本实验成功构建了以gfp和gpt为双重筛选标记的禽痘病毒通用转移载体pSY681- gfp-gpt,为禽流感重组活载体疫苗的研发提供了技术平台。获得了免疫原性较好的表达H9亚型禽流感病毒HA基因重组禽痘病毒,此重组活载体疫苗可为我国H9亚型禽流感的防制提供了新的疫苗储备。
Avian influenza(AI)is an infectious disease syndrome caused by type A influenza virus, which including highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). In recent years,the widespread H9 subtye avian influenza virus has caused great economic loss in poultry industry[0] in China.The recombinant virus is a potential vaccine to control avian influenza ,several different recombinant fowlpox virus(rFPV) expressing AIV HA gene demonstrated effective protection against homologous AIV challenge in chickens.
In this study fowlpox virus transfer vector pSY681- gfp-gpt with two reported genes(gfp and gpt) and two promoters ,P7.5(early) and P11(Later) in a back to back style was constructed. P11 initiated the expression of gfp gene and gpt gene, P7.5 was used for intiating exogenous gene. Two restriction sites NotI and Af1 II were inserted into downsteam of the early promoter P7.5, which were used for insertion of exogenous gene. HA gene of H9 subtype avian Influenza virus was inserted into designed site, then the recombinant vector pSY681-gfp-gpt-HA was transfected into CEF cells which infected with S-FPV-017 previously . Fowlpox virus recombinants were selected by gfp and gpt following several cycles purification.The results of PCR and Western-blot indicated that recombinant FPV could eficiently express HA gene of AIV in CEF.Twenty-one-days old SPF chickens were inoculated with the recombinant virus and challenged three weeks later with 106EID50 of LPAIV isolate per chicken.In order to evaluate the protection efficacy, throat and cloacal swabs were detected for virus existing in 3,5,7 days after challenge.HI test was also conducted before and after challenge.HI antibody could be dectected one week after inoculation and reached the level of 7log2 in three weeks. There was low virus shedding rate in the inoculation groups(15%) while 100% in control group. The high antibody level(HI test) and low virus shedding rate reflected that the recombinant fowlpox virus could produce satisfactary immune protection effect.
The successfully construction of the universal transfer vector pSY681- gfp-gpt with dual selection system(gpt and gfp) would greatly improve the development of live vector vaccine for AI.The recombinant virus rFPV -HA9 also could be a candidate vaccine for the control of Avian influenza.
本实验构建的禽痘病毒通用转移载体pSY681- gfp-gpt含有黄嘌呤鸟嘌呤磷酸转移酶(gfp)和绿色荧光蛋白(gpt)两个报告基因及背对的禽痘病毒晚期启动子P11和早期启动子P7.5,P11启动gfp和gpt两个基因,P7.5用于启动外源基因,在早期启动子P7.5下游引入NotI和Afl II两个酶切位点,用于外源基因的插入。将H9亚型禽流感病毒的HA基因插入到此载体,从而构建出表达HA基因的重组禽痘病毒转移载体pSY681- gfp-gpt-HA。应用TransIT?-LT1 Transfection Reagent将此转移载体转染已感染禽痘病毒S-FPV-017的鸡胚成纤维细胞,利用gfp和gpt同时进行双重筛选、数轮蚀斑纯化后获得了重组病毒rFPV -HA9。通过PCR、Western-blot鉴定,结果证明,rFPV -HA9能稳定表达AIV HA蛋白。用该重组病毒进行鸡翅刺种免疫21日龄SPF鸡,免疫3周后,每只鸡用106EID50的H9亚型LPAIV A/Chicken/Shanghai/10/01(H9N2)攻毒,分别于攻毒后第3、5、7天采集喉头及泄殖腔拭子进行病毒分离、滴定检测排毒情况,同时检测免疫后、攻毒前及攻毒后血清HI抗体、AGP抗体的动态变化,对重组病毒的免疫保护效果进行全面评估。结果,免疫组在免疫后一周就检测到了HI抗体,免疫后三周HI抗体水平达到了7log2以上,而且其AGP抗体均为阴性;攻毒后,免疫组在第三天只有15 %的鸡只在喉头有低水平的一过性的排毒,第五天全部停止排毒,其AGP抗体均为阴性。而对照组鸡只攻毒后第三天和第五天100%排毒,其AGP抗体均为阳性。通过Hl抗体水平和抑制排毒效力反应了本实验构建的重组禽痘病毒能够产生很好的免疫保护力。
总之,通过本实验成功构建了以gfp和gpt为双重筛选标记的禽痘病毒通用转移载体pSY681- gfp-gpt,为禽流感重组活载体疫苗的研发提供了技术平台。获得了免疫原性较好的表达H9亚型禽流感病毒HA基因重组禽痘病毒,此重组活载体疫苗可为我国H9亚型禽流感的防制提供了新的疫苗储备。
Avian influenza(AI)is an infectious disease syndrome caused by type A influenza virus, which including highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). In recent years,the widespread H9 subtye avian influenza virus has caused great economic loss in poultry industry[0] in China.The recombinant virus is a potential vaccine to control avian influenza ,several different recombinant fowlpox virus(rFPV) expressing AIV HA gene demonstrated effective protection against homologous AIV challenge in chickens.
In this study fowlpox virus transfer vector pSY681- gfp-gpt with two reported genes(gfp and gpt) and two promoters ,P7.5(early) and P11(Later) in a back to back style was constructed. P11 initiated the expression of gfp gene and gpt gene, P7.5 was used for intiating exogenous gene. Two restriction sites NotI and Af1 II were inserted into downsteam of the early promoter P7.5, which were used for insertion of exogenous gene. HA gene of H9 subtype avian Influenza virus was inserted into designed site, then the recombinant vector pSY681-gfp-gpt-HA was transfected into CEF cells which infected with S-FPV-017 previously . Fowlpox virus recombinants were selected by gfp and gpt following several cycles purification.The results of PCR and Western-blot indicated that recombinant FPV could eficiently express HA gene of AIV in CEF.Twenty-one-days old SPF chickens were inoculated with the recombinant virus and challenged three weeks later with 106EID50 of LPAIV isolate per chicken.In order to evaluate the protection efficacy, throat and cloacal swabs were detected for virus existing in 3,5,7 days after challenge.HI test was also conducted before and after challenge.HI antibody could be dectected one week after inoculation and reached the level of 7log2 in three weeks. There was low virus shedding rate in the inoculation groups(15%) while 100% in control group. The high antibody level(HI test) and low virus shedding rate reflected that the recombinant fowlpox virus could produce satisfactary immune protection effect.
The successfully construction of the universal transfer vector pSY681- gfp-gpt with dual selection system(gpt and gfp) would greatly improve the development of live vector vaccine for AI.The recombinant virus rFPV -HA9 also could be a candidate vaccine for the control of Avian influenza.
引文
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