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Construction, expression and purification of human cystatin C eucaryon expression vector

A technology of eukaryotic expression vector and expression vector, which is applied in the field of expression and purification of recombinant human cystatin C, which can solve the problems of poor antibody specificity, low sensitivity, and poor specificity of natural cystatin C

Inactive Publication Date: 2011-05-25
WUHAN LIFE ORIGIN BIOTECH LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] At present, there are many methods for the recombination and expression of human cystatin C, but all of them are expressed in the E. coli system. Another fact is that most of the commercially available cystatin C monoclonal antibodies are Antibodies have the disadvantages of poor specificity and low sensitivity
We believe that during the protein expression process using the prokaryotic system, due to the lack of the same expression environment as the natural cystatin C, the structure of the recombinant cystatin C is different from the natural cystatin C, resulting in the recognition of the prepared monoclonal antibody. Natural cystatin C has the disadvantages of poor specificity and low sensitivity

Method used

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  • Construction, expression and purification of human cystatin C eucaryon expression vector
  • Construction, expression and purification of human cystatin C eucaryon expression vector
  • Construction, expression and purification of human cystatin C eucaryon expression vector

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Embodiment 1

[0028] Construction, expression and purification of cystatin C eukaryotic expression vector

[0029] (1) Construction of cystatin C eukaryotic expression vector.

[0030] Primers were designed according to the sequence of cystatin C:

[0031] upstream primer ATCG GGATCC TCCAGTCCTGGCAAG

[0032] downstream primer C GGAATTC TTA ATGATGATGATGATGATG GGC GTCCTGACAGGT

[0033] Among them, a BamHI restriction site was introduced into the upstream primer, and an EcoRI restriction site and a 6×histidine tag (indicated by the underlined part) were introduced into the downstream primer. Carry out PCR amplification with 56 degrees as the annealing temperature. After the gel is recovered, it is digested, connected with the pcDNA3.1(+) vector digested with BamHI and EcoRI and transformed into Escherichia coli DH5a. After PCR identification, select positive recombinants to carry out Sequencing identification.

[0034] (2) Cystatin C expression in CHO cells

[0035] The correct clon...

Embodiment 2

[0037] Use Beijing Jiuqiang Cystatin C Detection Kit to measure the purified protein:

[0038] (1) First use the Mindray BS300 automatic biochemical analyzer to calibrate according to the method steps mentioned in the kit manual, and the working curve is shown in the appendix figure 2 .

[0039] (2) Dilute the purified protein to about 8.50 μg / ml with protein protection solution, and use the above curve to measure three times, and the average value of the result is 7.80 μg / ml.

[0040] (3) Use the protein protection solution to dilute the above-measured protein ratio to a series of values ​​(including the original concentration and 0 concentration of the protein protection solution), that is, 7.80 μg / ml, 7.02 μg / ml, 6.24 μg / ml, 5.46 μg / ml ml, 4.68 μg / ml, 3.90 μg / ml, 3.12 μg / ml, 2.34 μg / ml, 1.56 μg / ml, 0.78 μg / ml, 0 μg / ml.

[0041] (4) Then use a biochemical analyzer to measure the above concentration values ​​three times to get the average value, and the correlation coeffic...

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Abstract

The invention discloses a method for expressing and purifying recombinant human cystatin C proteins by using a eukaryon expression system. Because posttranslational modification and other mechanisms are lacked in a pronucleus expression system, the expressed and purified proteins have the defects of low activity and large structural difference as compared with the natural proteins, which results in that a cystatin C detection agent is difficult in the establishment process by using a monoclonal antibody prepared from Escherichia coli source recombinant cystatin C proteins. In the method, the defects of the pronucleus expression are overcome by utilizing the eukaryon expression system so as to obtain the high-activity recombinant human cystatin C protein, and a powerful basis is provided for obtaining a high-quality antibody.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method for expressing and purifying recombinant human cystatin C using a eukaryotic expression system. Background technique [0002] Cystatin C gene is a "housekeeping gene", which can be expressed in almost all nucleated cells and has no histological specificity, so the production rate of cystatin in the body is quite constant. Because cystatin is a low-molecular-weight protein that can be freely filtered by the glomerulus and reabsorbed and degraded in the proximal convoluted tubule, the kidney is the only organ that removes cystatin in the circulation, so the concentration of serum cystatin is mainly determined by The glomerular filtration rate (GFR) determines that cystatin is an ideal endogenous marker to reflect changes in GFR. [0003] At present, there are many methods for the recombination and expression of human cystatin C, but all of them are expressed in the E. coli system. Another...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/12C07K14/47C07K1/16
Inventor 华权高沈鹤霄
Owner WUHAN LIFE ORIGIN BIOTECH LTD
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