Method for expressing hABCB11 in insect cells sf9
An insect cell, sf9 technology, applied in the field of biological genetic engineering, to achieve the effect of good repeatability and high flexibility
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Embodiment l
[0031] 【 Embodiment 1] Construction of pFastBac1-hABCB11 expression vector
[0032] (1) Obtain the hABCB11 gene comprising BamHI and HindIII restriction sites at both ends:
[0033]According to the NCBI reference sequence information NM_003742.2, the sequence with a length of 4177bp was selected as the target sequence, a BamHI restriction site sequence was added at the 5' end of the target sequence and a HindIII restriction site sequence was added at the 3' end, the total sequence The length is 4189bp, and the detailed sequence information can be found in the attached table (SEQ ID No.5). Send the sequence information to a gene synthesis company to artificially synthesize the gene (Suzhou Jinweizhi Biotechnology Co., Ltd.).
[0034] BamHI restriction site sequence GGATCCAAGCTT HindIII restriction site sequence.
[0035] (2) The hABCB11 gene is cloned into the pFastBac1 vector:
[0036] Use BamHI and HindIII endonucleases (New England Biolabs Inc.) to perform double enzyme d...
Embodiment 2
[0044] [Example 2] Obtaining of recombinant baculovirus
[0045] Transforming pFastBac1-hABCB11 recombinant plasmid DNA into DH10Bac Escherichia coli competent cells (Yingwei Jieji (Shanghai) Trading Co., Ltd.) (which contains a baculovirus shuttle vector for short Bacmid, and a helper plasmid) to obtain the recombinant baculovirus shuttle vector Bacmid-hABCB11 inserted into the hABCB11 gene.
[0046] (1) Transformation of recombinant pFastBac1-hABCB11 into DH10Bac E. coli competent cells (transposition):
[0047] ①Take 1ug of the recombinant expression vector pFastBac1-hABCB11 and add 100uL DH10Bac Escherichia coli competent cells (Invitrogen (Shanghai) Trading Co., Ltd.), shake gently, put in ice water bath for 30 minutes, heat treatment at 42°C for 45 seconds, Then quickly remove to ice water for 2 minutes.
[0048] ② Add 900 ul of sterilized LB liquid medium to the tube, shake and incubate at 220 rpm at 37°C for 1 hour to allow transposition to occur.
[0049] ③Use the ...
Embodiment 3
[0059] [Example 3] Transfection of sf9 cells with recombinant baculoform shuttle vector virus Bacmid-hABCB11
[0060] (1) Transfect sf9 monolayer cells:
[0061] In each well of a six-well plate for cell culture, 9 × 10 5 SF9 cells were cultured overnight, and transfected when the cell density reached 60%-70%. Prepare the following solutions in sterile microcentrifuge tubes:
[0062] Solution A: 2 μg recombinant Bacmid-hABCB11 plasmid DNA plus 100 μL serum-free and antibiotic-free insect cell culture medium;
[0063] Solution B: 6 μL lipofectamine transfection reagent Cellfectin (Invitrogen (Shanghai) Trading Co., Ltd.) plus 100 μL serum-free and antibiotic-free insect cell culture medium;
[0064] Then gently mix liquid A and liquid B evenly, and place at room temperature for 15 minutes. At the same time, remove the culture liquid in each well of the six-well plate, wash once with serum-free and antibiotic-free insect cell culture medium, and discard the culture liquid. ...
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