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Method for constructing stable cell strain secreting expression protein

A technology of secretion expression and construction method, which is applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., which can solve the problems of heavy workload and difficult purification, and achieve low cost and low reagent cost , the effect of less impurity protein

Pending Publication Date: 2017-08-15
合肥知恩生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if a large amount of target protein needs to be expressed, a large amount of plasmid extraction, cell culture, transfection, broken cells are required to extract the total protein, and then purified, which requires a large workload and difficult purification.

Method used

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  • Method for constructing stable cell strain secreting expression protein
  • Method for constructing stable cell strain secreting expression protein
  • Method for constructing stable cell strain secreting expression protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of pCDH-S-His recombinant lentiviral vector

[0039] Plasmid pCDH-CMV-MCS-EF1-copGFP-T2A-Puro( figure 1 ) to perform double digestion with XbaI and EcoRI to recover the vector backbone. Such as figure 2 as shown,

[0040] Design and synthesize 2 oligonucleotide chains with the following sequences:

[0041] Forward: (5'→3') with XbaI sticky ends

[0042] CTAGAATGGAGACAGACACACTCCTGCTGTGGGTGCTGCTGCTCTGGGTCCCAGGTCCCACTGGCGACGGG (SEQ ID NO. 7);

[0043] Reverse: (5'→3') with EcoRI cohesive ends

[0044] AATTCCCGTCGCCAGTGGAGCCTGGGACCCAGAGCAGCAGCACCCACAGCAGGAGTGTGTCTGTCTCCATA (SEQ ID NO. 8).

[0045] Anneal the above two oligonucleotide chains, and connect them with the backbone of the carrier at 16°C with ligase, transform into E. coli overnight for 1 hour, and pick a single clone colony for overnight culture the next day, extract the plasmid and verify it by sequencing, and finally obtain the recombinant Vector pCDH-S.

[0046] The recombinant...

Embodiment 2

[0054] Example 2 Construction of pCDH-S-PEDF-His recombinant plasmid

[0055] Such as image 3 as shown,

[0056] Using the recombinant lentiviral vector pCDH-S-His as the backbone, the backbone part was recovered by double digestion with EcoRI and NotI.

[0057] Primers were designed to amplify the PEDF gene, and the primer sequences were as follows:

[0058] Upstream primers:

[0059] 5'-TTTGAATTCTATGCAGGCCCTGGTGCTA-3' (SEQ ID NO. 1);

[0060] Downstream primers:

[0061] 5'-TTTGCGGCCGCGGGGCCCCTGGGGTCCAGAA-3' (SEQ ID NO. 2);

[0062] Using human cDNA as a template, the PEDF gene was obtained by PCR reaction. The amplification program was pre-denaturation at 95°C for 4 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 20 seconds, 35 cycles, and finally extension at 72°C for 10 minutes.

[0063] After the PCR product was purified, it was digested with EcoRI and NotI, recovered by running the gel, and then ...

Embodiment 3

[0064] Example 3 Construction of pCDH-S-PLGF-His recombinant plasmid

[0065] Such as Figure 4 as shown,

[0066] Using the recombinant lentiviral vector pCDH-S-His as the backbone, the backbone part was recovered by double digestion with EcoRI and NotI.

[0067] Design primers to amplify the PLGF gene, and the primer sequences are as follows:

[0068] Upstream primers:

[0069] 5'-TTTGAATTCCCTGCCTGCTGTGCCCCCCCA-3' (SEQ ID NO.3);

[0070] Downstream primers:

[0071] 5'-TTTGCGGCCGCGCCTCCGGGGAACAGCAT-3' (SEQ ID NO.4);

[0072] Using human cDNA as a template, the PLGF gene was obtained by PCR reaction. The amplification program was pre-denaturation at 95°C for 4 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 20 seconds, 35 cycles, and finally extension at 72°C for 10 minutes.

[0073] After the PCR product was purified, it was digested with EcoRI and NotI, recovered by running the gel, and then ligated w...

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Abstract

The invention belongs to the field of biotechnology and particularly relates to a method for constructing a stable cell strain secreting an expression protein. The method comprises the following steps: with a recombinant lentiviral vector pCDH-S-His as a skeleton, constructing a target protein gene to the recombinant lentiviral vector pCDH-S-His to obtain a recombinant plasmid pCDH-S-P-His; co-transfecting the recombinant plasmid and helper plasmids pSPAX2 and pMD2.G with 293T cells by using a PEI transfection method to obtain a recombinant lentivirus; transducing the obtained lentivirus to the 293T cells, and performing puromycin screening to obtain a plurality of monoclonal cell strains; detecting the content of target protein in each cell supernatant by an Elisa method; determining a high-expression cell strain. Compared with a traditional plasmid transfection method, the method provided by the invention has the advantages of short experimental period, high positive rate and good stability of the cell strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for constructing a stable cell line that secretes and expresses proteins. Background technique [0002] The construction of eukaryotic expression plasmids, which can be transfected into 293T cells, can realize the transient expression of proteins, which is currently the most commonly used eukaryotic protein expression method. However, if a large amount of target protein needs to be expressed, a large amount of plasmid extraction, cell culture, transfection, broken cells are required to extract the total protein, and then purified, which is a heavy workload and difficult to purify. [0003] Simultaneous co-transfection of lentiviral expression vector and packaging plasmid into 293T cells can produce high-titer virus particles. Then, the virus acts on the host cell, so that the target gene can enter the host cell, undergo reverse transcription, and integrate into the genome, ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10
CPCC12N5/0636C12N15/86C12N2510/02C12N2740/15043C12N2800/107
Inventor 曹齐树李燕华刘宗梁张智李光伟
Owner 合肥知恩生物技术有限公司
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