Pseudomonas aeruginosa gene engineering vaccine candidate antigen PA5505 purification method

A technology of PA5505 and Pseudomonas aeruginosa, applied in genetic engineering, microbial-based methods, peptide preparation methods, etc., can solve the problems of no recombinant protein purification method research, unknown structure and function, etc., to achieve good results Immunoprotective effect, effect of high humoral immune response

Active Publication Date: 2019-03-19
重庆艾力彼生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The problem in the prior art is: PA5505 is a hypothetical protein with unknown structure and function, and there is no literature reporting on it, and there is no research on the purification method of the recombinant protein
The difficulty and significance of solving the above technical problems: PA5505 is a brand new protein with unclear physical and chemical properties, and there is no mature purification process for reference. It is necessary to explore the purification process of this protein based on experience

Method used

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  • Pseudomonas aeruginosa gene engineering vaccine candidate antigen PA5505 purification method
  • Pseudomonas aeruginosa gene engineering vaccine candidate antigen PA5505 purification method
  • Pseudomonas aeruginosa gene engineering vaccine candidate antigen PA5505 purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Construction of recombinant engineering bacteria pGEX-6p-2-PA5505 / XL-1blue

[0067] According to the coding sequence of PA5505, the upstream and downstream primers were designed, and the target sequence was obtained by PCR amplification. The target sequence and the pGEX-6p-2 vector were digested with BamH1 and Xho1 and ligated with T4 ligase to obtain the recombinant plasmid pGEX-6p- 2-PA5505( figure 2 It is the result of double enzyme digestion identification of the recombinant plasmid). The above-mentioned recombinant plasmid was transformed into competent Escherichia coli XL-1blue to obtain recombinant engineered bacteria pGEX-6p-2-PA5505 / XL-1 blue.

Embodiment 2

[0068] Example 2: Expression and enzyme digestion identification of PA5505

[0069] Take 200 μL of the pGEX-6p-2-PA5505 / XL-1 blue bacterial solution stored in the refrigerator at 4 °C and add it to 20 mL of LB medium containing Amp resistance for one activation. IPTG with a final concentration of 200 μM was placed in a shaker at 16°C for overnight induction. After the induction was over, the cells were collected by centrifugation at 5000 rpm for 10 minutes. After the cells were resuspended in 1.5 mL of PBS, the cells were ultrasonically lysed for 2 minutes (200 V), and the supernatant was collected. Combined with 40 μL of Glutathione Sepharose 4B (GE Company) gel beads (beads) used for binding GST fusion protein, the binding condition was 4 ° C for 3 h; after the binding was completed, unbound foreign proteins were eluted with PBS for 3 times, and then After resuspending the medium with 40 μL PBS, take 40 μL for electrophoresis. Add 5 μL of PreScissionprotease (PP enzyme, GE ...

Embodiment 3

[0070] Example 3 Purification process research of PA5505

[0071] By exploring the purification conditions of PA5505, the process flow of the protein purification was finally determined, such as figure 1 As shown, the specific operation is as follows.

[0072] 1. Autoclave, centrifuge

[0073] The Escherichia coli engineering bacteria expressing PA5505 were subjected to high-density fermentation, and the bacterial cells were collected by centrifugation for future use.

[0074] Take about 300g of bacteria, mix and suspend in PBS buffer according to the weight:volume ratio of 1:5, and pre-cool at 4°C.

[0075] 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.

[0076] 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 the broken ba...

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Abstract

The invention belongs to the technical field of biological pharmacy, and discloses a pseudomonas aeruginosa gene engineering vaccine candidate antigen PA5505 purification method. A DNA (deoxyribonucleic acid) sequence for encoding PA5505 protein activity function fragment is cloned to a pGEX-6p-2 vector by a gene engineering technology, escherichia coli recombinant bacteria pGEX-6p-2-PA5505 / XL-1 blue are built, and PA5505 proteins are acquired by inducible expression. Gene engineering bacteria expressing PA5505 are subjected to technologies such as high-pressure bacteria breaking, GST affinitychromatography, PP enzyme digestion, SP HP chromatography and Q HP chromatography to obtain high-purity vaccine candidate antigen PA5505. The purification method is simple in purification process, easy to amplify and good in repeatability, the acquired target proteins are high in purity, and animal experiments prove that a body can be effectively stimulated to generate high humoral immune response and good immune protection functions.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for purifying Pseudomonas aeruginosa gene engineering vaccine candidate antigen PA5505. Background technique [0002] PA is one of the most common clinical opportunistic pathogens, ranking the first among Gram-negative bacteria in clinical infection, and has become one of the pathogenic bacteria with the highest infection rate in ICU wards, burns, and war wounds in the world (Horino T et al., InternMed , 2012). PA infection can occur in any part and tissue of the human body, usually in burns or trauma, middle ear, cornea, urethra and respiratory tract, and can also cause endocarditis, gastroenteritis, empyema and even sepsis. The infection can be single or mixed, and the mechanism is complex. In recent years, the incidence of nosocomial infection, especially pulmonary infection, in PA has been increasing, and the mortality rate of severe infectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C07K14/21C07K1/22C07K1/16C12R1/19
CPCC07K14/21
Inventor 郭刚卢文根周璐杨念张娇娇
Owner 重庆艾力彼生物科技有限公司
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