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Method for expressing hABCG2 in insect cell sf9

A technology of insect cells and sf9, applied in the field of biogenetic engineering, achieves 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 hABCG2 in insect cells

Method used

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  • Method for expressing hABCG2 in insect cell sf9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0042] [Example 1] Construction of pFastBac1-hABCG2 expression vector

[0043] (1) Obtain the hABCG2 gene containing BamHI and HindIII restriction sites at both ends:

[0044] According to the NCBI reference sequence information , the sequence with a length of 2331bp was selected as the target sequence, and the BamHI restriction site sequence was added to the 5' end of the target sequence and the HindIII restriction site sequence was added to the 3' end. The total length of the sequence is 2343bp, 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 for artificial gene synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.).

[0045] (SEQ ID No. 5): BamHI restriction site sequence GGATCC AAGCTT HindIII restriction site sequence.

[0046] (2) The hABCG2 gene was cloned into the pFastBac1 vector:

[0047] The hABCG2 gene and pFastBac1 vector (English Weijieji (Shanghai) Trading Co., ...

Embodiment 2

[0055] [Example 2] Acquisition of recombinant baculovirus

[0056] The pFastBac1-hABCG2 recombinant plasmid DNA was transformed into DH10Bac Escherichia coli competent cells (Yingweijieji (Shanghai) Trading Co., Ltd.) (which contains a baculovirus shuttle vector referred to as Bacmid, and a helper plasmid) to obtain the insertion of the hABCG2 gene. The recombinant baculovirus shuttle vector Bacmid-hABCG2.

[0057] (1) Transformation of recombinant pFastBac1-hABCG2 into DH10Bac E. coli competent cells (transposition):

[0058] ①Take 1ug of recombinant expression vector pFastBac1-hABCG2, add 100uL DH10Bac E. coli competent cells (Invitrogen (Shanghai) Trading Co., Ltd.), shake and mix, put it in an ice-water bath for 30 minutes, and heat at 42°C for 45 seconds. Then quickly move to ice water for 2 minutes;

[0059] ②Add 900ul of sterilized LB liquid medium to the tube, shake and culture at 220rpm at 37℃ for 1 hour to make it transposition;

[0060] ③ Prepare a 10-fold seri...

Embodiment 3

[0070] [Example 3] Transfection of sf9 cells with recombinant rod-shaped shuttle vector virus Bacmid-hABCG2

[0071] (1) Transfection of sf9 monolayer cells:

[0072] Inoculate 9 × 10 cells with 2 mL of serum-free and antibiotic-free insect cell culture medium (Invitrogen (Shanghai) Trading Co., Ltd.) in each well of a six-well plate for cell culture. 5 SF9 cells were cultured overnight and transfected when the cell density reached 60% to 70%. Prepare the following solutions in sterile small centrifuge tubes:

[0073] Solution A: 2μg recombinant Bacmid-hABCG2 plus 100μL serum-free and antibiotic-free insect cell culture medium;

[0074] Solution B: 6 μL of liposome transfection reagent Cellfectin (Invitrogen (Shanghai) Trading Co., Ltd.) plus 100 μL of serum-free and antibiotic-free insect cell culture medium;

[0075] Then gently mix solution A and solution B, and leave at room temperature for 15-45 minutes. At the same time, remove the culture medium in each well of the...

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Abstract

The invention relates to a method for expressing hABCG2 in an insect cell sf9. The method comprises the following steps: cloning a human ABCG2 gene with BamHI and HindIII enzyme digestions at two ends into BamHI and HindIII sites of a pFastBac1 carrier respectively; converting an obtained recombinant plasmid pFastbac1-hABCG2 into a DH10Bac escherichia coli competent cell to obtain a recombinant baculovirus shuttle vector Bacmid-hABCG2 which is inserted into the hABCG2 gene; mediating the recombinant baculovirus shuttle vector Bacmid-hABCG2 with lipidosome to transfect an sf9 insect cell to obtain hABCG2 recombinant virus, and further culturing to obtain the expressed hABCG2 protein. According to the method disclosed by the invention, a novel way is provided for obtaining hABCG2 proteins on a large scale, and a working foundation is laid for the application and development of the hABCG2 proteins.

Description

technical field [0001] The invention relates to a method for expressing hABCG2 in insect cell sf9, which belongs to the field of biogenetic engineering. Background technique [0002] ABCG2 is the second protein member of the G family in the seven ABC family transporters, and 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. Immunohistochemistry shows that ABCG2 is mainly expressed in the cell membrane and cytoplasm, and has the functions of absorption, secretion, and excretion; at the same time, ABCG2 is an ATP-binding transporter, which can inhibit the absorption of certain exogenous substances in the digestive tract and participate in the formation of blood-brain and fetal blood barriers In addition, ABCG2 is also a new drug efflux pump related to tumor multidrug resistan...

Claims

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Application Information

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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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