[Baculovirus expression and establishment of the indirect ELISA for the HA gene of swine influenza virus H1 subtype].
The hemagglutinin (HA) gene fragment of swine influenza virus A/Swine/Guangdong/LM/05(H1N1) was amplified with HA gene specific primers and cloned into baculovirus transfer plasmid pFASTBacGP67B. The recombinant plasmid pFastBacGP67B-H1 was identified by restriction enzyme digestion and gene sequencing. Following the transformation of DH10Bac Escherichia coli component cells by pFastBacGP67B-H1, recombinant bacmids rBacmid-H1 were identified by blue/white selection and PCR analysis. Then recombinant baculovirus rBV-H1 was rescued by lipofectant reagent Cellfectin induced rBacmid-H1 DNA transfection of long-phage sf9 insect cells. The recombinant HA protein was characterized by hemagglutination test, western-blot and immunohistochemistry. An indirect enzyme-linked immunosorbent assay (ELISA) was assessed to detect in pigs IgG against H1 subtype SIV present in Inner Mongolia, Liaoning and Heilongjiang provinces. Positive was found in 31.15% (29 of 93) serum samples tested from swine reared in commercial herds. However, all irrelevant control sera tested were negative. We conclude, therefore, that ELISA performed with recombinant HA as coating antigen was a better tool for swine influenza surveillance in China.
Wan
Chunhe
C
Animal Influenza Laboratory of the Ministry of Agriculture, Harbin Veterinary Research Institute of CAAS, Harbin 150001, China. chunhewan@126.com
Liu
Ming
M
Liu
Chunguo
C
Zhang
Xiaoji
X
Yang
Tao
T
Liu
Dafei
D
Chen
Hao
H
Qi
Jinlong
J
Qiao
Chuanling
C
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007915:Lepidoptera; D009976:Orthomyxoviridae Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D013553:Swine Diseases / Q000175:diagnosis* / Q000276:immunology / Q000821:virology
D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus