新城疫病毒（dnv）hn基因真核表达重组质粒的构建.pdf

图表目录图1 NDV Lasota株HN的RT—PCR产物及其酶切鉴定?????????24 图2pcDKA3与Lasota株HN的RT—PCR双酶切回收产物???????24 图3载体pcDNA3结构简图????????????????????25 图4重组质粒pcDNA3一LHN的构建及酶切鉴定策略图???????·?”25 图5含有Lasota株HN基因的重组质粒的筛选???????????26 图6含有Lasota株HN基因的重组质粒的酶切鉴定?????????26 图7 NDV F48E9株}IN的RT—PCR产物???????????????27 图8pcDNA3与F48E9株HN的RT—PCR双酶切回收产物????????27 图9重组质粒pcDNA3~FHN的构建及酶切鉴定策略图?????????28 图10含有F48E9株HN基因的重组质粒的筛选???????????28 图ll重组质粒pcDNA3一FHN的双酶切鉴定??????????????29 图12重组质粒pcDNA3一LHNl插入片段5 7、3’端序列测定??????30 图13重组质粒DcDNA3一LHN2插入片段5’、3 7端序列测定??????30 图14重组质粒pcDNA3一LHNI插入片段全序列测定??????????3l 图15重组质粒pcDNA3一FHN插入片段全序列测定???????????32 图16 pcDNA3一FHN HN基因cDNA与genebank中F48E9 HN序列比较‘?一33 图17pcDNA3一LHN HN基因cDNA与genebank中Lasota tlN及 LAS/46HN序列比较????????????????????36 表1三类疫苗的比较??????????????????????6 新城疫病毒(NDV)HN基因真核袭达重组质粒的构建攘要应臻反转录一聚合酶链反应(RT—PCR)获褥两株新城疫瘸毒(NDV)盘凝索一神经氨酸酶(HN)熬因cDNA,然后定向插入真核表达质粒pcDNA3中, 构建了含HN基因的重组质粒。蓠先,将NDV Lasota株和F48E9株分别按种子9日龄SPF鸡胚,于 37℃孵化36h,置4"C12h屠收获鸡骚暴囊渡。投摆GenBank登袋黥这嚣拣藏毒豹}lN基蠢痔爱霸pcDNA3静多壳窿酶移j位患,分裂设诗秀对零|物Pl:5 ’。GTl’AAG一3 7,P2:5’一{3eTCl=A- TGGCTTCTC-3’和P3:5’G—.3 7,P4:5’TTGA— ‘。 HN 基霆eDNA。臻骚糖毫泳镰象显示,其太小均稳为l 700bp,与GenBank登录的大小一致。其次,将HN基因eDNA定向插入pcDNA3中。经双酶切鉴定,获得5个含HN攥因eDNA的重组质粒,其中Lasota株的4个(pcDNA3一LHNI、pcDNA3 一LHN2、pcDNA3一LHN3和pcDNA3-LHN4),F48E9株的1个(pcDNA3一FHN)。进一步测定重缝质数pcDNA3-LHNl孝pcDNA3一FttN孛骶基因片段鲍棱营酸序列,袭弱与GenBank登蒙的招应兹}{N基鞠序戮一致。综上所述,本研究成功地构建了含Lasota栋和F48E9株的HN蕊因eDNA 的真桉表达重组质粒,为新城疫新型核酸疫苗的实际应用奠定了潺础。焚键词:薪城疫病毒(NDV);HN基因;囊核表达重组质粒;壮J建 Construetion oftheEukaryotic Expression binant Plasmid Containing NDV—HN Gene Abstract Inthisstudy binant plasmids containing HN(hemagglutinin— neuraminidase)genes of NDV fNewcastle disease virus)were constructed, HN genes were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR)and were inserted directly into eukaryotic expression plasmid NDV strains,Lasota and F48E9,were propagated in 9-day-old embryonated specific-pathogen-free(SPF)eggs respectively for36hours at37。C, thenthesechicken embryos were pl