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Purification method in preparation of methicillin staphylococcus aureus-resistant recombinant genetic engineering vaccine candidate antigen I12C

A genetically engineered vaccine and methicillin-resistant technology, applied in the field of biotechnology and pharmaceuticals, can solve the problems of unfavorable vaccine industrial preparation, cumbersome methods, and low content, and achieve the effects of clear vaccine ingredients, controllable quality, and low cost

Active Publication Date: 2013-01-30
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the pathogenic factors of methicillin-resistant Staphylococcus aureus include dozens of capsular polysaccharides, ClfA, IsdB, enterotoxin, TSST-1, α-hemolysin, and coagulase, etc., and their contents are low, directly from the whole bacteria It is difficult to separate and purify the protective antigen, and the method is cumbersome, which is not conducive to the industrial preparation of vaccines

Method used

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  • Purification method in preparation of methicillin staphylococcus aureus-resistant recombinant genetic engineering vaccine candidate antigen I12C
  • Purification method in preparation of methicillin staphylococcus aureus-resistant recombinant genetic engineering vaccine candidate antigen I12C
  • Purification method in preparation of methicillin staphylococcus aureus-resistant recombinant genetic engineering vaccine candidate antigen I12C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Expression I 12 Construction of C engineering bacteria

[0055] Antigen I 12 The nucleotide sequence of C is shown in SEQ ID NO:1, and the amino acid sequence of its protein is shown in SEQ ID NO:2.

[0056] 1. Take out the MRSA-252 strain of methicillin-resistant Staphylococcus aureus from the freezer at -80°C and spread it on the special solid medium for MRSA-252, cultivate it overnight at 37°C, and then pick a single colony and inoculate it on MRSA- 252 special liquid culture medium for 8 hours, referring to the bacterial genome extraction kit (Shanghai Sangong) to extract the MRSA genome.

[0057] 2. Using the PCR method to self-synthesize template I 12 Amplification I 12 -Linker-gene fragment, the amplification steps are as follows:

[0058] 1) Design PCR primers P1 and P2, which are SEQ ID NO: 9-10, respectively, among them, P1 (5'-GCGGATCCATGGGCAGCGCACCAAACTCTCG-3') and P2 (5'-GCTTCTTTACTGCTGCTGCCACCGCCACCGGCATTGGCTTTAGTAAA-3').

[0059] 2) Using...

Embodiment 2

[0100] Example 2: Expression I 12 High-density fermentation of C engineering bacteria.

[0101] 1) Recovery, activation and identification of MRSA vaccine engineered bacteria for fermentation

[0102] (1) Recovery of engineered strains of MRSA vaccine

[0103] Take 100 μl of strains preserved at -70°C in 20% glycerol, inoculate them on a plate containing A+LB solid medium, and incubate overnight at 37°C. After the colonies grow, store them at 4°C.

[0104] (2) Activation of seed bacteria

[0105] Pick a single colony with uniform shape and size and inoculate it in a medicine bottle containing 5ml of A+LB medium, culture at 37°C and 200r / min for 5-7h, and the OD600 reaches 0.6-0.8, become activated bacteria, and store at 4°C.

[0106] (3) Identification of seed fungus

[0107] The activated bacteria were taken for morphological detection, Gram staining detection, antibiotic resistance detection, biochemical reaction detection, and bacterial species identification.

[0108...

Embodiment 3

[0124] Example 3: Expression I 12 Autoclaving and centrifugation of C engineering bacteria

[0125] The recombinant double-subunit genetic engineering protein I constructed by the applicant to express soluble methicillin-resistant Staphylococcus aureus 12 The Escherichia coli engineering bacteria of C, through high-density fermentation, the expression rate of the target protein is 13%, and the bacteria are collected by centrifugation for later use.

[0126] 200-500 g of bacterial cells were mixed with PBS (10-20 mM, pH 7.0-7.5) buffer solution according to the weight: volume ratio of 1:10, and pre-cooled at 4°C.

[0127] High-pressure homogenizer: Use distilled water to flush the pipeline of the high-pressure homogenizer, and turn on the low-temperature circulation system to pre-cool to 1-4°C for later use.

[0128] Add the pre-cooled suspended bacteria liquid to a high-pressure homogenizer, maintain the pressure at 60-80Mpa to break the bacteria 3-5 times, take a smear of t...

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Abstract

The invention relates to a purification method in preparation of methicillin staphylococcus aureus-resistant recombinant di-subunit genetic engineering vaccine candidate antigen I12C, which comprises the following steps of: collecting prepared antigen; and carrying out high-pressure bacterium breaking and centrifuging, precipitating ammonium sulfate step by step to purify the prepared I12C antigen according to the sequential combination, i.e., the GST (glutathione s transferase) affinity chromatography technology, the PP (propene polymer) digestion technology, the buffer solution replacement technology, the Resource Q chromatography technology and the gel filtration chromatography technology. The purification method provided by the invention is simple and direct, easy to amplify, and good in repeatability, so that the obtained target protein is high in purity; the animal experiment proves that the purified antigen can effectively stimulate the organism to generate higher humoral immune response and have good immune protection function; and the purity of the antigen I12C purified by the method provided by the invention is more than or equal to 99%.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and relates to a candidate antigen I of methicillin-resistant Staphylococcus aureus recombinant genetic engineering vaccine 12 Purification method in C preparation. Background technique [0002] Systemic inflammatory response syndrome and sepsis / septic shock caused by war trauma and infection are one of the main causes of death of war trauma patients, and the fatality rate is as high as 50-80%. So far, there is no effective prevention and treatment drug. Among them, Staphylococcus aureus (Staphylococcus aureus, SA) is one of the most common sources of infection. With the long-term and extensive use of antibiotics, the problem of drug resistance of Staphylococcus aureus has become increasingly prominent. Methicillin-resistant Staphylococcus aureus (MRSA) is a typical representative, because of its wide transmission route, it is easy to cause outbreaks It is popular, and because of its s...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K1/36C07K1/30C07K1/22C07K1/16C12N15/70C12R1/19
Inventor 顾江邹全明曾浩董衍东樊绍文卢陆
Owner CHENGDU OLYMVAX BIOPHARM
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