H3N2亚型猪流感病毒HA1、HA2基因的原核表达及抗原性分析

doi:10.16431/j.cnki.1671-7236.2016.10.037

Abstract

Abstract:

Hemagglutinin protein plays an important role in disease prevention and treatment of the swine influenza virus (SIV).In order to clone and express subtype H3N2 SIV A/swine/Henan/1/2010 (H3N2) HA1 and HA2 genes with chicken embryo allantoic fluid containing the H3N2 SIV after extraction of RNA.The HA1 gene and HA2 gene were amplified from the total RNA using RT-PCR and they were inserted into prokaryotic expression vector pET-28a (+) to construct recombinant expression vector.Then the vector was transformed and expressed in E.coli BL21 (DE3) pLyS.Then the bacteria were induced by IPTG and their lysates were analyzed by SDS-PAGE and Western blotting,respectively.The monoclonal antibody 1C10,which was specific to HA protein,was used as primary antibody in Western blotting analysis.It was found that the expressed recombinant HA1 protein was 34.8 ku and recombinant HA2 protein was 23.2 ku analyzed by SDS-PAGE.As demonstrated by Western blotting,this HA1 expressed products showed the capacity of reacting with monoclonal antibody 1C10.All the results suggested that the expressed HA1 and HA2 proteins of H3N2 influenza virus would be very helpful in the development of rapid diagnosis method and vaccine development,which would facilitate further study on the function of HA at the same time.

Key words: influenza A virus subtype H3N2; HA1 gene; HA2 gene; prokaryotic expression

CLC Number:

S852.65⁺9.5

SONG Yu-hui, WANG Xiang-yu, WANG Jie-qiong, PAN Meng-meng, ZHANG Jian, LIU Lin, LI Xin-sheng. Prokaryotic Expression and Antigenic Analysis of H3N2 Swine Influenza Virus HA1 and HA2 Genes[J]. , 2016, 43(10): 2762-2767.

References

[1] 刘大飞,杨涛,刘明,等.H3N2亚型猪流感病毒HA基因序列测定及抗原性分析[J].动物医学进展,2008,29(3):5-9.
[2] 刘天强.H3N2亚型猪流感病毒HA1蛋白间接ELISA方法的建立及NP蛋白单克隆抗体的制备[D].北京:中国农业科学院,2008.
[3] 张烨,于在江,黄捷勤,等.流感病毒H3N2血凝素基因和神经氨酸酶基因在大肠杆菌中的表达[J].生物技术通报,2011,1:213-217.
[4] 朱春平,李燕,吴亚丽,等.H3N2亚型SIV核蛋白基因的克隆及在大肠杆菌中的表达[J].西北农林科技大学学报(自然科学版),2015,3:1-6.
[5] 陈欣,张乐萃,卢春晓,等.H9N2亚型禽流感病毒辽宁分离株HA1蛋白的原核表达[J].动物医学进展,2013,34(10):89-91.
[6] Iba Y,Fujii Y,Ohshima N,et al.Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses[J].Journal of Virology,2014,88(13):7130-7144.
[7] 邵京京,杨靖,戴军,等.Ag85A-HA2原核表达载体的构建及其抗甲型流感病毒免疫效果的研究[J].四川大学学报(医学版),2014,45(4):547-551.
[8] 周雪媚,霍惠玲,张飚,等.禽流感病毒HA1基因的克隆及原核表达[J].动物医学进展,2006,27(1):66-69.
[9] Ekiert D C,Bhabha G,Elsliger M-A,et al.Antibody recognition of a highly conserved influenza virus epitope[J].Science,2009,324(5924):246-251.
[10] Gigant B,Barbey-Martin C,Bizebard T,et al.A neutralizing antibody Fab-influenza haemagglutinin complex with an unprecedented 2:1 stoichiometry:Characterization and crystallization[J].Acta Crystallographica Section D,2000,56(8):1067-1069.
[11] Baras B,de Waal L,Stittelaar K J,et al.Pandemic H1N1 vaccine requires the use of an adjuvant to protect against challenge in naïve ferrets[J].Vaccine,2011,29(11):2120-2126.
[12] 文雪霞,包红梅,孙佳善,等.抗禽流感病毒NS1蛋白单克隆抗体的制备及其抗原表位的初步分析[J].中国预防兽医学报,2013,35(5):414-418.
[13] Blanchfield K,Kamal R P,Tzeng W P,et al.Recombinant influenza H7 hemagglutinins induce lower neutralizing antibody titers in mice than do seasonal hemagglutinins[J].Influenza and Other Respiratory Viruses,2014,8(6):628-635.
[14] Strengell M,Ikonen N,Ziegler T,et al.Antibody responses against influenza A(H1N1) pdm09 virus after sequential vaccination with pandemic and seasonal influenza vaccines in finnish healthcare professionals[J].Influenza and Other Respiratory Viruses,2013,7(3):431-438.
[15] 庞耀珊,谢芝勋,谢丽基,等.H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析[J].基因组学与应用生物学,2012,31(2):109-116.
[16] Yamashita A,Kawashita N,Kubota-Koketsu R,et al.Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2,H1N1 and H5N1:Implication for human monoclonal antibody recognition[J].Biochemical and Biophysical Research Communications,2010,393(4):614-618.

Prokaryotic Expression and Antigenic Analysis of H3N2 Swine Influenza Virus HA1 and HA2 Genes

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