Recombinant human adiponectin expression vector, vector construction method and expression method

A technology of expression vector and construction method, applied in vector, nucleic acid vector, recombinant DNA technology and other directions, can solve the problems of inconsistent evaluation of adiponectin effect and difference of adiponectin effect, and achieves high repeatability, good versatility, and solution. complex effects

Pending Publication Date: 2021-04-13
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there are several problems in the current research on adiponectin: 1. There may be differences in the effects of

Method used

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  • Recombinant human adiponectin expression vector, vector construction method and expression method
  • Recombinant human adiponectin expression vector, vector construction method and expression method
  • Recombinant human adiponectin expression vector, vector construction method and expression method

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Embodiment 1

[0080] Embodiment 1 Construction of recombinant human adiponectin expression vector

[0081] Synthesize the cDNA sequence shown in SEQ ID No.1;

[0082] Using the cDNA sequence as a template, it was amplified by PCR. The total reaction system of polymerase chain reaction is: template cDNA 1 μL, forward primer 3 μL, reverse primer 3 μL, 5×PrimeStar Buffer 6 μL, dNTPS (2.5mM each) 0.5 μL, Taq DNA Polymerase 0.5 μL, add ddH 2 0 to 30 μL. The reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 25 cycles; extension at 72°C for 5 min.

[0083] After the polymerase chain reaction product was analyzed by 10g / L agarose gel electrophoresis, the band at about 748bp was quickly taken for gel tapping. For electrophoresis results see figure 2 , figure 2 There is an obvious band at 748bp, which is the target fragment. The target fragment was recovered by agarose electrophoresis and then d...

Embodiment 2

[0088] Example 2 Expression of recombinant human adiponectin fusion protein in hamster ovary cells

[0089] CRISPR technology knocked out the endogenous glutamine synthetase gene of CHO-K1 cells, took CHO-K1 cells (Invitrogen) in logarithmic growth phase, digested with trypsin, resuspended in PBS solution, and adjusted its density About 1×10 7 cells / ml. The above cell suspension was mixed with 20ug ADIP-FC-GS plasmid, and placed on ice for 5min. Transfer the cell suspension to a pre-cooled electric shock cup (165-2081, Bio-rad, capacity 0.4ml, gap 0.4cm), adjust the electroporator to square wave mode, voltage 280v, time 20ms, electric shock once, total discharge Time 20ms. Transfer the cells to a 10cm culture dish and add 10ml DMEM complete medium. Then place them in a carbon dioxide incubator for overnight cultivation. On the second day, the medium was replaced with a screening medium containing 50 uMMSX (M5379, SIGMA, USA), and the culture was continued for about 14 day...

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Abstract

The invention relates to the technical field of biology, in particular to a recombinant human adiponectin expression vector, a vector construction method and an expression method. The recombinant human adiponectin expression vector comprises a nucleotide sequence as shown in SEQ ID No. 1 and an Fc-GS plasmid. The recombinant human adiponectin expression vector construction method comprises the following steps of carrying out codon optimization according to an amino acid sequence of human adiponectin to obtain a cDNA sequence of human adiponectin, wherein the cDNA sequence of the human adiponectin is as shown in SEQ ID No. 1; designing a primer, amplifying the cDNA sequence, and recovering a target fragment after identification; and connecting the target fragment to the Fc-GS plasmid to obtain the recombinant human adiponectin expression vector. When the recombinant human adiponectin expression vector is transfected into hamster ovary cells, recombinant human adiponectin-Fc fusion protein can be stably expressed; and the method is simple, convenient and efficient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant human adiponectin expression vector, a vector construction method and an expression method. Background technique [0002] Adiponectin (ADIP) is an adipocyte factor specifically expressed and secreted into plasma by adipocytes. Its monomer molecular weight is about 30kD, and it is named Acrp30. Under normal circumstances, the expression level of adiponectin in the body is stable, the concentration is about 3-30 μg / mL, accounting for 0.01% of the total protein content in plasma. [0003] The tissue-specific expression of adiponectin messenger RNA was 100-fold higher than that in other tissues during adipocyte differentiation. The human adiponectin gene is located on chromosome 3q27, with a total length of 16Kbp, consisting of three exons and two introns, including 244 amino acids, divided by function, from the amino terminal to the carboxyl terminal: signal peptide doma...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/12C07K19/00
CPCC12N15/85C12N15/66C07K14/47C12N2800/22C07K2319/30
Inventor 龚燕平李春霖刘敏燕卢艳慧闫双通孙般若李婷
Owner GENERAL HOSPITAL OF PLA
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