Recombinant protein A efficiently combined with IgG (Immunoglobulin G) and construction method of engineering bacterium thereof

A technology of recombinant protein and engineering bacteria, which is applied in the fields of molecular biology and genetic engineering, can solve the problems of increased risk of protein A isolation, poor extraction efficiency of protein A, and difficulty in large-scale extraction, so as to increase production efficiency and work Safety, simple extraction method, and the effect of increasing extraction efficiency

Inactive Publication Date: 2012-03-14
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the method of directly extracting protein A from the surface of Staphylococcus aureus is mainly used to obtain protein A. The extraction efficiency of protein A by this method is not good. Because the protein A content on the cell wall of Staphylococcus aureus is small, it is difficult to extract in large quantities, and gold Staphylococcus aureus is a pathogenic bacterium, directly isolating protein A from the bacterium will increase the risk of work and reduce the safety of the product

Method used

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  • Recombinant protein A efficiently combined with IgG (Immunoglobulin G) and construction method of engineering bacterium thereof
  • Recombinant protein A efficiently combined with IgG (Immunoglobulin G) and construction method of engineering bacterium thereof
  • Recombinant protein A efficiently combined with IgG (Immunoglobulin G) and construction method of engineering bacterium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Design of primers for recombinant protein A antibody binding region

[0020] According to the gene sequence published by NCBI, the primer P was designed using DNAMAN software. 1 and P 2 .

[0021] P 1 : 5'-ACCGCCATGGTATTGAAAAAGAAAAACATT-3'Noc I

[0022] P 2 : 5'-ACCGGGATCCTTAACATAGTTCGCGACGACGTCC-3'Bam H I

[0023] The part in italics in the primer is the restriction site, in the primer P 2 A cysteine ​​codon and a stop codon were added. The purpose of designing primers based on the IgG antibody binding region of protein A is to effectively reduce the length of the peptide chain and reduce the steric hindrance effect when binding the medium and IgG. A cysteine ​​is designed at the C-terminus of protein A, the purpose is to combine the activation medium with the -SH of cysteine, and improve the adsorption capacity for IgG.

Embodiment 2

[0024] Embodiment 2: Construction of recombinant plasmid pMD18-T-spa

[0025] [1] Chromosomal DNA extracted from Staphylococcus aureus as a template, extraction method: pick a single colony from a fresh Staphylococcus aureus plate with an inoculation loop and inoculate it in LB liquid medium, shake overnight at 37°C, and take out the next day Centrifuge 400μl of the bacterial solution at 8000r / min for 3min, discard the supernatant, wash the cells once with 0.5ml TE, discard the supernatant by centrifugation, suspend the cells sufficiently with 0.5ml TE, add 50μl of 10mg / ml lysozyme solution, mix well, 37 ℃ water bath for 2-3 hours, add 100μl 10% SDS, mix well, keep warm at 37℃ for 30min, add 50μl 10mg / ml proteinase K, react at 65℃ for 3h, mix once every half hour during this period, add an equal volume of saturated phenol, mix Evenly, centrifuge at 8000r / min for 5min, transfer the supernatant to another centrifuge tube, add an equal volume of saturated phenol to the supernatan...

Embodiment 3

[0028] Embodiment 3: Construction of recombinant plasmid pET-28a-spa

[0029] Construct the recombinant vector pET-28a-spa, double digest the plasmid extracted in Example 2 [3] and plasmid pET-28a with BamH I and Noc I, use the gel recovery box to recover and connect, connection system: target gene Digested product 7.5 μl, pET-28a digested product 0.5 μl, T4 DNA ligase buffer 1 μl, T4 DNA ligase 1 μl, ligated overnight at 16°C. The ligated recombinant plasmid pET-28a-spa was transformed into competent E.coil BL21, the transformation method was referred to Example 2[3], and positive colonies were picked with a Kanamycin plate (Kana+LB). The plasmid was extracted after culturing overnight on a shaker at 37°C, added glycerol after enzyme digestion was verified to be correct, and stored in a -20°C refrigerator for later use.

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Abstract

The invention discloses a recombinant protein A efficiently combined with IgG (Immunoglobulin G) and a construction method of an engineering bacterium thereof. The method comprises the following steps of: designing a recombinant protein A primer for IgG antibody combining areas E, D, A, B, C of a staphylococcal protein A (SPA) according to a gene sequence announced by the NCBI (National Center For Biotechnology Information), and adding a cysteine codon into an end C primer; amplifying by taking the genome of a staphylococcus aureus as a template to obtain a gene (SPA) of an encoding recombinant protein A antibody combining area, connecting the gene (SPA) with a plasmid pMD18-T, transforming competent E.coil JM109 to obtain a large quantity of recombinant plasmids pMD18-T-spa, storing the gene and increasing the copy numbers of the gene; and extracting the recombinant plasmids, performing double digestion on the recombinant plasmids and a plasmid pET-28a by using Noc I and BamH I, connecting a recombinant plasmid pET-28a-spa, transforming competent E.coil BL21, adding IPTG (Isopropyl beta-D-Thiogalactoside) of which the finial concentration is 1mmol/L for inducing expression at the temperature 30 DEG C, and identifying whether the expression is successful by using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electropheresis).

Description

technical field [0001] The invention discloses a recombinant protein A efficiently binding to IgG and a method for constructing genetically engineered bacteria thereof, belonging to the fields of molecular biology and genetic engineering. technical background [0002] Protein A, Staphylococcus aureus protein A (SPA), is a cell wall protein, and more than 90% of the cell walls of Staphylococcus aureus contain this protein, but different strains contain different amounts of protein A. Since the protein A molecule does not contain cystine and cysteine, there is no disulfide bond in the protein A molecule. Someone has done research on the stability of protein A, and found that protein A has good stability, with 4mol / L urea, thiocyanate hydrochloric acid, 6mol / L guanidine hydrochloride and acidic conditions of pH2.5, and heating and boiling does not affect its activity. [0003] Protein A is a single-chain polypeptide that is covalently bound to cell wall peptidoglycan and is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63C12N1/21C07K14/31C07K1/22C07K16/00C12R1/19
Inventor 夏海锋吴璞强金雄华饶志明
Owner JIANGNAN UNIV
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