Method for quickly constructing IBV (Avian Infectious Bronchitis Virus) reverse genetic strain
A bronchitis and reverse genetic technology, applied in the field of coronavirus reverse genetics, can solve the problems of transcript heterogeneity, coronavirus cannot be stably preserved, and the efficiency of enzymatic ligation is low, achieving high positive clone rate and improving rescue Efficiency and simple operation
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[0056] The chicken infectious bronchitis virus vaccine strain H120 used in the present invention is preserved by the virus seed bank of this laboratory; the passaged cell BHK-21 used is preserved by the cell bank of this laboratory.
[0057] The cloning vector pBeloBAC11 and the Escherichia coli DH10B strain were donated by Professor Huang Yaowei, School of Animal Science, Zhejiang University.
[0058] RNA extraction reagents, PCR high-fidelity enzymes, homologous recombination kits, and site-directed mutagenesis kits were purchased from Vazyme Company, reverse transcription kits were purchased from Thermo Company, restriction enzymes BamHI and XhoI were purchased from Takara Company, and transfection Reagent jetPRIME was purchased from Dakwei Biotechnology Co., Ltd.
[0059] The primers were synthesized by Sangon Bioengineering Co., Ltd., and the sequence determination was completed by Boshang Biotechnology Co., Ltd.
[0060] 1. Construction strategy of full-length cDNA of I...
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