A DNA fragment and its application in constructing recombinant influenza virus expressing red fluorescent protein gene
A red fluorescent protein, influenza virus technology, applied in the field of molecular biology
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Embodiment 1
[0047] Embodiment 1, the rescue of the H9N2 recombinant virus carrying mCherry gene
[0048] 1.1 Construction of NS gene full-length infectious clone carrying mCherry gene
[0049] Extract the parent virus strain (influenza virus A / chicken / Guangdong / V / 2008 (H9N2), referred to as V K627 ), design primers to amplify the gene sequences encoding NS1 protein and NEP protein (without stop codon), and insert the mCherry fluorescent gene behind the NS1 gene, which uses PTV that can be recognized and cleaved by proteases between the gene and the NEP gene -1 2A sequence connection, see the specific construction figure 1 ; wherein primers Aar I-V-NS1-F and V-NS1-mCherry-R amplify the sequence (NS1 gene sequence) encoding NS1 protein; mCherry-2A sequence is artificially synthesized by Jinweizhi Company; I-V-2A-NEP-R amplifies the gene sequence of NEP protein (NEP gene sequence), and inserts Aar I restriction sites in the upstream and downstream respectively. Primer sequences (5'-3') ar...
Embodiment 2
[0109] Example 2: V K627 - Identification and application of mCherry recombinant virus
[0110] 2.1 Identification of recombinant virus NS full-length gene and fluorescent gene mCherry
[0111] Recombinant virus V of generations P1 to P4 were extracted with the total RNA rapid extraction kit of Feijie Biotech Co., Ltd. K627 -The total RNA of mCherry was reversed using MLV reverse transcriptase (Takara code No.2641A) to obtain cDNA. The specific reverse transcription reaction system is as follows:
[0112] RTase M-MLV(RNase H-)(200U / μL) 1μL 5×M-MLV-Buffer 12μL dNTP Mixture (10mmol) 12μL RNase Inhibitor (40U / μL) 2μL Uni12primer (10pmol) 3μL RNA template 30μL total capacity 60μL
[0113] Design NS full-length identification primers (SEQ ID NO:25 and SEQ ID NO:26) and mCherry fluorescent gene identification primers (SEQ ID NO:27 and SEQ ID NO:28), the PCR reaction system is as follows:
[0114] 2×Premix Ex-Taq ...
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