摘要
目的 了解我国内地从猪中分离到H9N2亚型毒株HA和NA基因来源及它们使猪致病的原因。方法 用PCR扩增目的基因 ,与PGEM T EasyVector 4℃过夜连接 ,重组质粒转化DH 10 β细菌 ,筛选阳性菌落 ,酶切鉴定 ,测序。然后 ,进行进化树分析。结果 两株猪H9N2毒株HA蛋白分子上第 2 2 6位上氨基酸为L ,这与从人和猪所分离出的H9N2毒株相同 ,其连接肽属对禽致病的毒株 ,但它们的序列为R L S R ,而不是R S S R ;其NA蛋白茎区第 6 2～ 6 4位存在掉失 ,这与A Shaoguan 4 0 8 98,A Swine HongKong 9 98及A Duck HongKong y 2 80 97(H9N2 )毒株相同 ;HA与NA基因进化树分析表明 ,两株猪H9N2毒株的HA基因接近于A Chicken HongKong G2 3 97和A Chicken HongKong G9 97,而NA基因接近于A Shaoguan 4 0 8 98毒株。结论 两株猪H9N2亚型毒株的HA和NA基因可能性最大来自禽H9N2毒株。由于其HA蛋白分子上连接肽氨基酸序列发生替换 ,可能造成了它们对猪具有致病性。禽H9N2毒株NA蛋白茎区氨基酸掉失 ,造成了它们能直接感染猪。
Objective To understand the origin of HA and NA genes of swine influenza A (H9N2) viruses isolated from pigs in the mainland of China and on basis of these to reveal the pathogenecity of them in pigs. Methods The target gene was amplified by PCR,the PCR product was ligated with P GEM-T Easy Vector (Promega company,U S A) at 4℃,the recombined plasmid was transferred into DH-10β bacteria; positive colonies were selected and identified then digested with restriction enzyme.Afterwards,the nucleotide seguence was determined.Finally,phylogenetic analysis of the seguencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares. Results Two strains of swine influenza A(H9N2) virus isolated in the mainland had an amino acid residue,leucine (L) at position 226 (H3 numbering) on HA protein molecule found in H9N2 viruses isolated either in pigs or humans previously;the amino acid sequence at HA connecting peptide of isolates possessed R-L-S-R,whereas the other H9N2 viruses with virulence in poultry had R-S-S-R at HA connecting peptide. The two pig H9N2 isolates shared the same three-amino-acids deletion in the NA stalk at 62-64 position foumd in A/Shaoguan/408/98 and A/Swine/Hong Kong/9/98,as well as A/Duck/Hong Kong /Y280/97(H9N2) viruses. The analysis of the phylogenetic tree indicated that the HA and NA genes of new isolates were closely related to those of A/Chicken/Hong Kong/G23/97 and A/Chickon/Hong Kong/G9/97 and A/Shaoguan/408/98 viruses,respectively. Conclusion The HA and NA genes of swine influenza A(H9N2) viruses isolated in the mainland of China probably wese derived from those of avian influenza A(H9N2) virus. The occurrence of substitution of amino acid sequence at HA connecting peptide,could result in the H9N2 virus from non pathogenic to pathogenic in pigs. However,avian influenza A(H9N2) virus had deletion in the stalk of the NA that resulted in host range transmission.Therefore they could infect pigs directly.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2004年第1期7-11,共5页
Chinese Journal of Experimental and Clinical Virology
