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Preparation, renaturation and preservation method of VacA recombinant protein

A technology of recombinant protein and preservation method, which is applied in the field of preparation, renaturation and preservation of VacA recombinant protein, which can solve the problems of difficult purification, difficulty in interpreting and analyzing proteins, and heavy workload, and achieve the effect of reducing cell volume

Pending Publication Date: 2021-06-22
THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the natural VacA extracted from Hp has low yield, heavy workload and difficult purification; the crude pure liquid of VacA toxin collected from the supernatant of Hp secretion is complex and diverse, and it is difficult to explain and analyze the protein function; while the traditional VacA recombinant protein has many Inactive, it needs to be activated with HCl in vitro to become biologically active

Method used

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  • Preparation, renaturation and preservation method of VacA recombinant protein
  • Preparation, renaturation and preservation method of VacA recombinant protein
  • Preparation, renaturation and preservation method of VacA recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of VacA recombinant expression vector

[0043]Using the vcaA gene sequence (VacA toxin mature fragment, the sequence of expressing the 34th-854th amino acid) of the H.pylori standard strain (strain J99 / ATCC 700824) as a template to synthesize mutation primers, primers at both ends of the gene, the primer sequence is as SEQ ID NO.1 and SEQ ID NO.2. The sequence SEQ ID NO.1 contains the recognition site of the restriction endonuclease Nde I, and the sequence SEQ ID NO.2 contains the recognition site of the restriction endonuclease Xho I. PCR amplification was carried out under the following conditions: 96°C for 5 min of pre-denaturation; 96°C for 30 seconds, 57°C for 30 seconds, 72°C for 1 minute and 20 seconds, and 72°C for 5 minutes (25 cycles).

[0044] After the PCR product was digested with restriction enzymes Nde I and Xho I, it was inserted into the expression vector plasmid pET41b (Novagen) containing a C-terminal histidine tag (8His tag); the PCR pr...

Embodiment 2

[0047] Expression and purification of VacA recombinant protein

[0048] The recombined plasmid was transformed into Escherichia coli BL21(DE3) and coated with K + Plates and incubated overnight at 37°C. A single colony was then inoculated into 50 ml of LB medium containing kanamycin (50 μg / ml) and the colonies were grown overnight at 37° C. in a shaker at 200 rpm. Take 100ul and transfer it to containing K + The cells were cultured in 5ml LB of 200 rpm and continued to be cultured at 37°C and 200rpm to observe the cell growth density. When the cell proliferation and growth curve reached OD=1.2 at 600nm, 0.5mM IPTG was added, and under different conditions (20°C for 20h, 37°C for induction 4h, 15°C induction for 16h) protein expression was induced at 200rpm. After induction, the bacteria were resuspended, and the expression was identified by SDS-PAGE and Western blot.

[0049] Collect the bacterial liquid obtained from the above culture, discard the supernatant after centri...

Embodiment 3

[0054] The VacA recombinant protein purified from inclusion bodies was renatured, and the buffer shown in Table 1 was selected. The recombinant protein was renatured in the buffer shown in Table 1, and the renaturation analysis of the protein was determined by the solubility of the protein (when the protein was dissolved in the buffer, if a precipitate appeared, it indicated that the renaturation failed, and if it was completely dissolved, it indicated that the renaturation failed. Sexual success), or according to the results of SDS-PAGE and Western-blot analysis (the lysate was collected, and the protein content was further analyzed by SDS-PAGE and Western-blot).

[0055] Table 1 Selection of renaturation buffer solution

[0056]

[0057]

[0058] The renatured VacA recombinant protein was collected, passed through a dialysis membrane with a molecular weight cut-off of 14 kDa for 4 hours, and then dialyzed with the same buffer mentioned above for 16 hours, and finally d...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a preparation, renaturation and preservation method of VacA recombinant protein. The method comprises the following steps: acquiring a VacA gene sequence, and constructing a VacA recombinant expression vector after PCR amplification; transforming the expression vector into Escherichia coli, performing multiplication culture, and adding an inducer to induce protein expression to obtain bacterial liquid; centrifuging the bacterial liquid, taking a bacterial precipitate, and carrying out ultrasonic lysis on the bacterial precipitate by adopting a lysis buffer solution to obtain a cell lysate; purifying the cell lysate to obtain a VacA recombinant protein; the VacA recombinant protein is subjected to dialysis renaturation and preservation with an acetic acid buffer solution with the pH being 2.9. The high-activity VacA recombinant protein can be obtained through the preparation, renaturation and preservation method, and cell apoptosis can be induced.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the preparation, refolding and preservation methods of VacA recombinant protein. Background technique [0002] VacA toxin is one of the most important virulence factors of Hp, and VacA protein plays a very important role in the pathogenesis of chronic gastritis-cancer. [0003] At present, there is a lack of a unified high-efficiency expression system of VacA toxin, which hinders the basic research based on the structure and function of VacA. Studies have shown that the natural VacA extracted from Hp has low yield, heavy workload and difficult purification; the crude pure liquid of VacA toxin collected from the supernatant of Hp secretion is complex and diverse, and it is difficult to explain and analyze the protein function; while the traditional VacA recombinant protein has many It is inactive and needs to be activated with HCl in vitro to become biologically active. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P21/02C07K14/195C07K1/14C07K1/22C07K1/34C07K1/36C07K1/113
CPCC12N15/70C07K14/195
Inventor 王芬袁玲芝肖士郎蔡婷陈冰
Owner THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV
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