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Purification method of Pseudomonas aeruginosa vaccine recombinant protein reSBP

A technology of Pseudomonas aeruginosa and a purification method, which is applied in the field of purification of the recombinant protein reSBP of Pseudomonas aeruginosa vaccine, can solve the problems of inability to meet industrial application requirements, increase the cost of production auxiliary materials and operation steps, increase the cost of protein and the like

Active Publication Date: 2020-04-28
重庆艾力彼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To sum up, the problem in the prior art is that the key to vaccine research is to find antigens with good immunogenicity and immune protection effect.
It is well known in the art that the bioactive filler required for GST affinity chromatography is very expensive and has a short service life, which greatly increases the cost of protein production and purification. In addition, this purification step also requires enzyme cleavage with PP enzyme, which increases the cost of production auxiliary materials and operating steps reduce the recovery rate of the target protein, so it has not been seen that GST chromatography technology has been applied to the industrial production of vaccine proteins and other biopharmaceuticals
Therefore, the existing purification scheme of reSBP protein cannot meet the needs of industrial application because of the GST affinity chromatography step, which severely limits the application of reSBP as a vaccine candidate antigen

Method used

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  • Purification method of Pseudomonas aeruginosa vaccine recombinant protein reSBP
  • Purification method of Pseudomonas aeruginosa vaccine recombinant protein reSBP
  • Purification method of Pseudomonas aeruginosa vaccine recombinant protein reSBP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Synthesis and subcloning of genes

[0032] Introduce the Ndel restriction site at the 5' of the reSBP coding sequence, introduce the stop codon TGA and the Xhol restriction site at the 3', connect the sequence with the pET30a vector through the Ndel and Xhol restriction sites, and connect the connected sequence vector Transformed into Escherichia coli BL21 to obtain pET30a-reSBP / BL21.

[0033] The DNA sequence of reSBP was synthesized, and the connection between the sequence and pET30a was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0034] The DNA sequence of reSBP is shown in SEQ ID NO:1:

[0035] GCCGAGTCCCTCACCGTCGCGGCCACCCCGGTGCCGCACGCGGAGATCCTCAACGTGGTCAA GCCGCTGCTGGCCAAGGAAGGCGTGGACCTGAAGATCAAGGAGTTCACCGACTACGTGCAGCCGAA CGTGCAGGTCTCGGAAAAGCGCCTGGACGCCAACTTCTTCCAGCACCAGCCGTACCTCGATGAGTT CAACAAGGCCAAGGGCACCGACCTGGTCGCCGTGACCGGCGTACACATCGAGCCGCTGGGCGCCTA CTCGAGCAAGTACAAGAAGCTCGACGAACTGCCTTCCGGCGCTACCGTGGTGATTCCCAACGACGC CACCAACGGCGGCCGCGCCCT...

Embodiment 2

[0042] Example 2 Purification of recombinant fusion protein reSBP

[0043] 1. Large-scale cultivation of pET30a-reSBP / BL21

[0044] Take 100 μL of pET30a-reSBP / BL21 bacterial solution stored in a refrigerator at 4°C and add it to 20 mL of LB medium containing Kan+ resistance for primary activation. After culturing at 200 rpm for 12 hours at 37°C, take 10 mL of the primary activated bacterial liquid and add it to 4000 mL Contains Kan + Perform secondary activation in resistant LB medium, culture at 37°C for 3-4 hours until OD600 is 0.6-0.8, add 200 μL IPTG (final concentration: 100 μM) and place in a shaker at 16°C overnight for induction, then centrifuge at 6000 rpm for 10 minutes to collect bacteria body.

[0045] 2. Autoclave, centrifuge

[0046] About 20g of bacteria was dissolved in PBS (10mM, pH7.0-7.5) buffer solution (take 1 bag of PBS dry powder (1L / bag) and add 1000ml of grade I water to dissolve completely), mix and suspend according to the weight:volume ratio of ...

Embodiment 3

[0061] Example 3: Immunization of Animals

[0062] 1) Fifteen SPF grade BALB / C female mice aged 6-8 weeks were divided into experimental group, adjuvant control group and negative control group, with 5 mice in each group, purchased from Beijing Huafukang Company.

[0063] 2) For the first immunization, dilute the reSBP antigen with PBS and add Al(OH) at a concentration of 1mg / mL 3 ; Use a No. 5 half-gauge needle to inject into the muscles of both thighs. The injection volume of each BALB / C mouse in the experimental group was 100 μL, containing 50 μg of reSBP and Al(OH) 3 100μg; the injection volume of each BALB / C mouse in the adjuvant control group was 100μL, containing Al(OH) 3 100μg; each BALB / C mouse in the negative control group was injected with 100μL PBS, without protein and Al(OH) 3 adjuvant.

[0064] 3) The second immunization, the second immunization was carried out on the 14th day, the immune components were the same as above, the injection amount of protein ant...

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Abstract

The invention discloses a purification method of a Pseudomonas aeruginosa vaccine recombinant protein reSBP. The reSBP protein is obtained by recombinant fusion of active functional fragments of Pseudomonas aeruginosa antigen molecules, and expression of Escherichia coli genetically-engineered bacteria. The genetically-engineered bacteria are subjected to technologies of high-pressure bacterium breaking, SP FF chromatography, Phenyl HP chromatography, G 25 chromatography and the like so as to obtain the high-purity Pseudomonas aeruginosa (PA) recombinant genetically-engineered protein reSBP. Compared with existing purification schemes, the purification process of the invention is simple, avoids the use of GST affinity chromatography, greatly reduces the cost, and is easier for industrial amplification. The obtained target protein has high purity. Animal tests prove that the obtained protein can effectively stimulate an organism to generate high humoral immune response and a good immuneprotection effect.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a method for purifying recombinant protein reSBP of Pseudomonas aeruginosa vaccine. Background technique [0002] Pseudomonas aeruginosa (PA) is the most common pathogen in nosocomial infections such as burns, war trauma, and mechanically ventilated patients, and can cause severe pneumonia, pulmonary failure, sepsis and even death. WHO listed PA as the second in the "New Antibiotic Research, Discovery and Global Priority List of Antibiotic-Resistant Bacteria" released in 2017, which shows that the health problems caused by the prevalence of PA infection are very serious. Due to the multi-drug resistance or pan-drug resistance of PA to antibiotics, the clinical treatment effect is very limited. The data showed that the total separation rate of PA in 2016 was 8.69%, especially the separation rate of PA in mechanical ventilation pneumonia was as high as 22.9%. The annual resistan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/21C07K1/16C07K1/14C12N15/70
CPCC07K14/21C12N15/70Y02A50/30
Inventor 冯强郭刚张欣卢文根张娇娇杨念罗莉熊蜂
Owner 重庆艾力彼生物科技有限公司