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Method for expressing hABCC2 in insect cell sf9

An insect cell, sf9 technology, applied in the field of biological genetic engineering, to achieve the effect of good repeatability and high flexibility

Inactive Publication Date: 2015-07-01
苏州杰诺曼博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no related method to express hABCC2 in insect cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0031] [Example 1] Construction of pFastBac1-hABCC2 expression vector

[0032] (1) Obtain the hABCC2 gene comprising BamHI and HindIII restriction sites at both ends:

[0033] According to the NCBI reference sequence information NM_000392.3, the sequence with a length of 4838bp 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 4850bp, 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] (SEQ ID No.5): BamHI restriction site sequence GGATCCAAGCTT HindIII restriction site sequence.

[0035] (2) The hABCC2 gene is cloned into the pFastBac1 vector:

[0036] Use BamHI and HindIII endonucleases (New England Biolabs Inc.) to perform double ...

Embodiment 2

[0044] [Example 2] Obtaining recombinant baculovirus

[0045] Transforming pFastBac1-hABCC2 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-hABCC2 inserted into the hABCC2 gene.

[0046] (1) Transformation of recombinant pFastBac1-hABCC2 into DH10Bac E. coli competent cells (transposition):

[0047] ①Take 1ug recombinant expression vector pFastBac1-hABCC2 and add 100uLDH10Bac Escherichia coli competent cells (Yingwei Jieji (Shanghai) Trading Co., Ltd.) , shake gently to mix, put in ice water bath for 30 minutes, heat treatment at 42°C for 45 seconds, then quickly move 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 LB ...

Embodiment 3

[0059] [Example 3] Transfection of sf9 Cells with Recombinant Baculate Shuttle Virus Bacmid-hABCC2

[0060] (1) Transfect sf9 monolayer cells:

[0061] Serum-free, antibiotic-free insect cell culture medium at 2 mL per well in a six-well plate for cell culture (Yingwei Jieji (Shanghai) Trading Co., Ltd.) Inoculate 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-hABCC2 plasmid DNA plus 100 μL serum-free and antibiotic-free insect cell culture medium;

[0063] Solution B: 6μL liposome transfection reagent Cellfectin (Yingwei Jieji (Shanghai) Trading Co., Ltd.) Add 100 μL of 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, was...

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Abstract

The invention relates to a method for expressing hABCC2 in an insect cell sf9. The method comprises the following steps: cloning a human ABCC2 gene containing BamHI and HindIII restriction enzyme cutting sites at two ends respectively into BamHI and HindIII sites of a pFastBac1 vector to obtain a recombinant plasmid pFastBac1-hABCC2; transforming a DH10Bac competent escherichia coli cell with the obtained recombinant plasmid pFastBac1-hABCC2 to obtain a recombinant baculovirus shuttle vector Bacmid-hABCC2; transfecting the insect cell sf9 with the recombinant baculovirus shuttle vector Bacmid-hABCC2 in a liposome mediated way so as to obtain a recombinant virus expressing hABCC2; and further culturing the recombinant virus to express hABCC2 protein. The method disclosed by the invention provides a new way for acquiring a large quantity of the hABCC2 protein and also lays a foundation for application and development of the hABCC2 protein.

Description

technical field [0001] The invention relates to a method for expressing hABCC2 in insect cell sf9, which belongs to the field of biogenetic engineering. Background technique [0002] ABCC2 is one of the protein members of the C family of the ABC seven family transporters. It is expressed in human normal cells and tumor tissues, such as small intestinal epithelial apical membrane, placental syncytiotrophoblast, intestinal tubular membrane, and brain microvascular epithelial cells , breast lobules, and breast cancer cells. The main function of ABC protein is the transmembrane transport of small molecular substances and polypeptide molecules. The transmembrane region changes shape to allow passage of certain molecules. Immunohistochemistry shows that ABCC2 is mainly expressed in the cell membrane and cytoplasm, not only has the functions of absorption, secretion and excretion, but also has physiological functions such as inhibiting the absorption of certain exogenous substanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/12C07K14/47
Inventor 谢伟胜张科之
Owner 苏州杰诺曼博生物科技有限公司
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