Recombined Newcastle disease heat-resisting vaccine strain for expressing H5 subtype avian influenza virus truncated HA protein and preparation method
An avian influenza virus and vaccine strain technology, applied in the field of molecular biology, can solve the problem of lack of heat resistance, and achieve the effects of saving storage and transportation costs, reducing dependence, and significantly heat resistance.
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[0025] The first step, construction and identification of recombinant full-length plasmid pTS-HA1 / H5
[0026] 1.1 PCR amplification of HA1 gene of H5 subtype avian influenza virus
[0027] H5N1 avian influenza virus A / chicken / Hubei / 489 / 2004 strain (this virus strain has been reported in the literature, Luo Mengcheng, Prokaryotic expression and antibody preparation of H5N1 subtype avian influenza virus nucleoprotein NP, Chinese Journal of Zoonoses, 2008) proliferated on 9-11-day-old chicken embryos, and harvested chicken embryo allantoic fluid 5 days after inoculation. The harvested allantoic fluid was subjected to RT-PCR amplification of the HA1 gene, and the amplification primers were: upstream primer 5'- AAGCTTGCCACCATG GAGAAAATAGTGCTTCTTCTTGC-3', downstream primer 5'- ATCGGGGCACTCCGA TTCTACCCGTATTTTTCTTAATTATTATCCTCTCTTTTTTCTTCTTCTC-3' (the underlined part is the complementary sequence used for In-fusion cloning connection). The target band detected by agarose gel elec...
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