To control of H5N6 subtype highly pathogenic avian inlfuenza viruses(HPAIVs)with antigenic changes(clade 2.3.4.6),and the potential pandemic threat to poultry,a reassortant virus,named rC6,was constructed using reverse genetics in the study. The virus contained modified HA and NA genes of an epidemic strain A/Chicken/SC/6/2014 with the internal genes derived from A/Puerto Rico/8/34(PR8). The reassortant virus was attenuated by removal of the multi-basic amino acid motif in the HA gene by mutation and deletion(from PLRERRRKR to PQRETR),thus resulting in a candidate vaccine virus. Immune efficacy tests showed that the inactivated rC6 recombinant vaccine could induce high levels of hemagglutination inhibition antibody. Challenge tests showed that rC6 recombinant inactivated vaccines provided SPF chickens with 100%protection against homologous and heterologous H5N6 virus,and Re-5 vaccine against clade 2.3.4 viruses only provided about 10%protection. The rC6 recombinant vaccine candidate strains using reverse genetics technology provided a beneficial attempt for prevention and control of the virus of clade 2.3.4.6.%为应对抗原性已发生变异的第2.3.4.6分支H5N6亚型禽流感病毒（AIV）引发的潜在流行，本研究利用反向遗传操作技术，以A/Puerto Rico/8/34（PR8）的内部基因为骨架，以H5N6亚型AIV A/Chicken/SC/6/2014（C6）的基因组为模板，经RT-PCR扩增其HA及NA基因，并对HA基因进行分子修饰，去除与H5亚型AIV致病力有关的HA蛋白裂解位点处的多个碱性氨基酸，使其获得低致病性AIV的分子特征（即将-PLRERRRKR-突变为-PQRETR-），成功构建了H5N6亚型AIV疫苗候选株rC6。免疫效力试验表明，rC6重组灭活疫苗可以诱导产生高水平的血凝抑制抗体。免疫攻毒保护试验表明，rC6重组灭活疫苗可以提供SPF鸡抵抗同源和异源H5N6病毒100%的保护，而针对第2.3.4分支的Re-5疫苗仅能提供大约10%的保护。利用反向遗传技术构建的rC6重组疫苗候选株为该分支病毒的防控提供了有益的尝试。
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