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Expression vector for recombinant human keratin 19 antigen active fragments, and application thereof

A technology for expressing vectors and active fragments, applied in the field of genetic engineering, can solve the problems of low antigen activity, poor stability, and inability to reach natural extracts of recombinant protein.

Active Publication Date: 2010-03-03
上海裕隆生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a few companies also use gene recombination to express CK19 antigen in vitro (such as the CK19 recombinant protein encoding the full-length CK191-401aa gene produced by China-funded Hanmai Company), but due to the high mismatch rate, it can be obtained by ordinary PCR. It is difficult to encode the full-length gene of CK19; and the recombinant protein antigen activity expressed by constructing the expression vector based on this full-length gene is not high, can not reach the level of natural extracts, and has poor stability

Method used

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  • Expression vector for recombinant human keratin 19 antigen active fragments, and application thereof
  • Expression vector for recombinant human keratin 19 antigen active fragments, and application thereof
  • Expression vector for recombinant human keratin 19 antigen active fragments, and application thereof

Examples

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Embodiment 1

[0041] Example 1, the construction of human recombinant keratin 19 antigen active fragment expression vector pTrc-CKS-CK19-1 is as follows figure 1 As shown, the construction process of the vector pTrc-CKS-CK19-1 includes the following steps:

[0042] 1. Cloning of gene CK19-1 encoding active fragment of CK19 antigen

[0043] 1. Using the human cDNA library purchased from MegaMan as a template, design a pair of primers according to the nucleotide sequence to be intercepted. The sequences of the primers are as follows:

[0044] Primer 1: 5' ggatccgaca tgcgaagcca atatg3'

[0045] Primer 2: 5'gaattcgtga tcttcctgtc cctcg3'

[0046] The primer 1 sequence adds a BamH I restriction site to the 5' end of the truncated CK19 gene fragment CK19-1, and the primer 2 sequence adds an EcoR I restriction site to the truncated CK19 gene fragment CK19-13' end. The PCR operation was carried out in strict accordance with the standard operating procedures. In a 50 μl PCR system, 1 μl of cDNA, 1...

Embodiment 2

[0065] Example 2, Induced Expression of Human Recombinant Keratin 19 Antigen Active Fragment

[0066] 1. Small amount induced expression and identification of active fragment of human recombinant keratin 19 antigen

[0067] Medium:

[0068] 2×YT medium (g / L): 10 g of yeast extract, 16 g of peptone, 5 g of NaCl, containing 50 μg / ml Amp.

[0069] Inducer: 1mM IPTG (isopropyl-β-D-thiogalactoside)

[0070] The recombinant strain pTrc-CKS-CK19-1 / TOP10 was inoculated in 5ml of 2×YT medium at a ratio of 1:100, cultivated at 37°C and 250rpm for 3.5hrs, and 1ml of bacterial liquid was taken as a blank sample, and the remaining bacterial liquid was added to a final concentration of 1mM IPTG (isopropyl-β-D-thiogalactoside) was used to induce expression, and cultured at 28°C and 250rpm for 4hr.

[0071] Take the samples before and after induction, and centrifuge at 7000rpm for 6min to collect the bacteria. Lysis buffer (50mM NaH 2 PO 4 , 300mMNaCl, pH8.0) resuspended bacteria, and t...

Embodiment 3

[0074] Example 3, Purification of Human Recombinant Keratin 19 Antigen Active Fragment

[0075] , Pretreatment of expression products

[0076] Lysis buffer (50mM NaH 2 PO 4 , 300mM NaCl, pH8.0) ultrasonic crushing, the solution became clear after crushing, centrifuged at 14000rpm for 15min, and the supernatant was taken.

[0077] 2. Purification of expression products

[0078] The supernatant was passed through High-Affinity Ni-NTA (nickel ion affinity column, purchased from Jinsite Technology Co., Ltd.), and 10 column volumes of Wash Buffer (50mM NaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH8.0) to wash impurities, and then use Elution Buffer (50mM NaH 2 PO 4 , 300mM NaCl, 250mM imidazole, pH8.0) to elute the target protein.

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Abstract

The invention discloses an expression vector for recombinant human keratin 19 antigen active fragments, and application thereof, which aims to provide the construction of a recombinant expression vector cloning recombinant keratin 19 high-antigen-activity gene fragments, as well as a method for producing and preparing the recombinant keratin 19 antigen active fragments by utilizing the expressionvector. The expression vector codes a nucleotide sequence in a keratin 19 antigen cDNA full-length sequence, and the sequence contains a gene coding two monoclonal antibody BM19-21 and KS19-1 bindingepitopes. If the expression vector is cloned into an escherichia coli pTrc-CKS expression vector, efficient soluble expression can be realized, and the yield can be up to 15 mg / L. The activity and stability of the expression vector are detected and reach an applicable level. The in vitro efficient expression of CK19 not only improves the disadvantage that the CK19 is low in yield, high in production cost and poor in stability, but also greatly improves the specificity of obtained protein antigens.

Description

1. Technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an expression vector of an antigenic active fragment of human recombinant keratin 19 and a method for expressing an antigen by using the recombinant vector. 2. Technical background [0002] Keratin is the intermediate filament of the cell body, with a central highly conserved α-helical rod region surrounded by highly variable amino- and carboxy-terminal regions. DNA sequence studies of keratin have shown that keratin polypeptides are products of gene transcription rather than proteolytic fragments. According to the relative molecular mass and isoelectric point of keratin, 20 bands of human epithelial cytokeratin can be separated by two-dimensional electrophoresis, named CK1-20 respectively, with pI value of 5.2-7.8 and molecular mass of 40000-60000 , These keratins are divided into two types: type I has an acidic isoelectric point, and the molecular ma...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N15/12C12P21/04C12R1/19
Inventor 穆海东汪宁梅刘纲任韧
Owner 上海裕隆生物科技有限公司