腮腺炎疫苗株Jeryl Lynn 1反向遗传系统的建立

Construction of a reverse genetics system of mumps virus vaccine strain Jeryl Lynn 1

目的建立腮腺炎疫苗株Jeryl Lynn 1(JL1)反向遗传系统,获得单一成分的拯救病毒JL1R。方法将JL1全长cDNA基因序列分为7个片段(F1~F7),在F7片段的3′端引入丁型肝炎病毒核酶序列(hepatitis D virus ribozyme,HdvRZ),分段合成后分别插入载体pT7-MCS3中,根据每段重叠区域的酶切位点拼接,构建全长表达质粒pT7-MCS3-MUVJL1(HdvRz);同时构建表达腮腺炎病毒(mumps virus,MuV)核蛋白(NP)、磷蛋白(P)、聚合酶蛋白(L)的3个辅助质粒;将全长表达质粒与3个辅助质粒及表达T7RNA聚合酶的质粒pCDIBP-T7RNAP共同电转染Vero细胞。对获得的病毒JL1R进行测序、病毒滴度及中和抗体测定。结果拯救病毒JL1R基因组SH及HN片段与疫苗株JL1对应片段的理论序列一致;JL1R经Vero细胞连续传代培养2~10代病毒滴度均在6 lgCCID50/mL以上;JL1R与疫苗株S79诱发抗MuV抗体滴度接近。结论成功构建了MuV疫苗株JL1的反向遗传操作系统,获得的单一成分拯救病毒JL1R可作为腮腺炎疫苗株的备选株,解决了S79疫苗生产中毒种及疫苗株的成分、纯度和批间一致性等问题,进一步提高了国内腮腺炎疫苗的免疫效果。

Objective To construct a reverse genetics system of mumps virus vaccine strain Jeryl Lynn 1(JL1)and to obtain a rescued virus strain JL1R with a single composition.Methods The full-length cDNA of JL1 was divided into seven fragments,F1~F7,based on unique restriction enzyme cleavage sites.A hepatitis D virus ribozyme sequence was introduced to the 3’-terminus of F7,while the seven fragments were synthesized and inserted into vector pT7-MCS3 respectively,then connected together in accordance with the enzyme cleavage sites in two overlapping regions to generate a full-length expression vector pT7-MCS3-MUVJL1(HdvRz).Meanwhile,three helper plasmids coding for viral proteins NP,P and L were constructed.Electroporation was used to transfect Vero cells with pT7-MCS3-MUVJL1(HdvRz)and helper plasmids.The obtained JL1R was sequenced and determined for titer and neutralizing antibody.Results The SH and HN sequences of genome of JL1R were consistent with those of the corresponding sequences of vaccine strain JL1.After subculture in Vero cells,the titers of JL1R of passages 2~10(P2~P10)reached more than 6 lgCCID50/mL.However,the titers of antibody against MuV induced by JL1R and vaccine strain S79 were in agreement.Conclusion A reverse genetics system was successfully constructed for mumps virus vaccine strain JL1.The single-component rescued virus JL1R can be used as an alternative to the current vaccine strain S79 for mumps vaccine production,ensuring the composition,purity and consistency of the virus seed and vaccine strain during S79 vaccine production and further improving the immune effect of domestic mumps vaccine.