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Fermentation and purification process of helicobacter pylori LuxS hexamer recombinant protein

A technology of Helicobacter pylori and fermentation process, applied in the directions of fermentation, recombinant DNA technology, bacterial peptides, etc., can solve the problems of complicated process, difficult stability of oral Helicobacter pylori vaccine, high cost, and achieve simple and pure fermentation and purification process. High, immune effect

Pending Publication Date: 2021-07-23
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the Helicobacter pylori vaccine developed by physical mixing or intermolecular fusion has immune protection effect, the stability of the oral H. Intramolecular adjuvant effect, and most of them are precipitated and expressed, purified and prepared into vaccines through inclusion body renaturation, the process is complicated and the cost is high

Method used

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  • Fermentation and purification process of helicobacter pylori LuxS hexamer recombinant protein
  • Fermentation and purification process of helicobacter pylori LuxS hexamer recombinant protein
  • Fermentation and purification process of helicobacter pylori LuxS hexamer recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 LTA1-LuxS-LTA1-LTA2-LTB gene construction

[0070] (1) LTA1, LuxS, LTA2, LTB gene cloning and connection

[0071] 1. LTA1, LTA2, and LTB genes are derived from LT, and LT is derived from whole gene synthesis (Shanghai Jierui Bioengineering Co., Ltd.).

[0072] 2. The LuxS gene is derived from Helicobacter pylori SS1 strain.

[0073] 3. According to the principle of primer design, design corresponding primers and add enzyme cutting sites. Primer sequences are shown in Table 1:

[0074] Table 1

[0075]

[0076]

[0077] 4. LTA1, LuxS, LTA2, LTB gene connection

[0078] (1) The high-fidelity PCR method is used to amplify the gene sequence, and the high-fidelity system and procedures refer to the instructions.

[0079] (2) High-fidelity PCR was performed with LT as template and F-82 and R-82a as primers.

[0080] (3) High-fidelity PCR was performed with the whole genome of Helicobacter pylori SS1 strain as template and F-83a and R-83a as primers.

[...

Embodiment 2

[0113] Embodiment 2, the fermentation process of pET28a-LTA1-LuxS-LTA1-LTA2-LTB Escherichia coli engineering bacteria

[0114] (1) Basic procedure of fermentation

[0115] 1. Inoculation and culture of pET28a-LTA1-LuxS-LTA1-LTA2-LTB Escherichia coli

[0116] The constructed pET28a-LTA1-luxs-LTA1-LTA2-LTB Escherichia coli engineered bacteria (culture 4) was taken out from the -80°C ultra-low temperature refrigerator, and the engineered bacteria were inoculated in LB liquid medium containing 0.001% kanamycin Medium, 37°C, 220rpm constant temperature culture overnight.

[0117] 2. pET28a-LTA1-luxs-LTA1-LTA2-LTB Escherichia coli transferred to 10L fermenter culture

[0118] Take out the engineered bacteria cultivated overnight and inoculate in a 10L fermenter with a 10% inoculation ratio, the fermentation medium is TB medium, and the inoculation volume is 5L. Incubate for 8 hours at 37°C with a dissolved oxygen concentration of 30%. The composition of TB medium is: potassium d...

Embodiment 3

[0152] Embodiment 3 Purification process of LuxS hexamer recombinant protein

[0153] (1) Basic conditions for purification

[0154] Instrument system: AKTAprimer (GE)

[0155] Filler: D-Galactose

[0156] Purification column model: 7510081 (Bio-Rad)

[0157] Packing volume: 2mL

[0158] Buffer C1 composition: disodium edetate 0.11%, sodium chloride 1.17%, Tris 0.30%, glycerol 1.00%.

[0159] Buffer C2 composition: 0.11% disodium edetate, 1.17% sodium chloride, 0.30% Tris, 1.00% glycerol, 4.50% D-galactose.

[0160] (2) Purification process

[0161] 1. Bacteria

[0162] Take 10 g of the bacteria obtained from the fermentation, add buffer C1 at a mass (g): volume (mL) ratio of 1:20, and use a shearer to shear and suspend the buffer containing the bacteria at 4°C.

[0163] Use RO water to flush the high-pressure homogenizer (AH-1500, ATS Industrial Systems Co., Ltd.) pipeline. Turn on the low-temperature refrigeration system for pre-cooling and reserve. Add the pre-cool...

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Abstract

The invention discloses a fermentation and purification process of helicobacter pylori LuxS hexamer recombinant protein, and relates to the technical field of biology. According to the invention, a constructed expression vector containing an LTA1-LuxS-LTA1-LTA2-LTB gene is transferred into Escherichia coli engineering bacteria to carry out amplification culture, and then induction and purification are performed so as to determine the optimal induction and purification process. According to the invention, the LuxS hexamer contains the monomer antigen LTA1-LuxS-LTA1-LTA2 and an intramolecular adjuvant LT (B) 5 pentamer, the LuxS hexamer recombinant protein can be subjected to supernatant expression through fermentation, the LuxS hexamer recombinant protein can be subjected to one-step affinity chromatography purification, and the provided LuxS hexamer recombinant protein can effectively stimulate organisms to cause the protective immune response. The LuxS hexamer recombinant protein has both an adjuvant and an antigen, and a new method is provided for development of a helicobacter pylori oral mucosal vaccine.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a fermentation and purification process of Helicobacter pylori LuxS hexamer recombinant protein. Background technique [0002] Helicobacter pylori infection accounts for more than half of the global population. In some infected persons, it can cause chronic gastritis and gastric ulcer. At the same time, the occurrence of gastric mucosa-associated lymphoid tissue lymphoma (MALT) is closely related to Helicobacter pylori infection. The World Health Organization has listed Helicobacter pylori as a class of carcinogens. [0003] Helicobacter pylori is colonized in the gastric mucosa, and most of the currently studied vaccines are oral immunizations, and the adjuvants are mostly LT, CT, LT (B) 5 、CT(B) 5 . The above-mentioned adjuvants and the candidate antigens of Helicobacter pylori are usually mixed physically for oral immunization, or recombinant proteins are obtain...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12P21/02C07K19/00C07K1/14C07K1/22A61K39/106A61K39/39A61P31/04G01N33/68G01N33/569
CPCC12N15/70C07K14/195A61K39/105A61K39/39A61P31/04G01N33/6854G01N33/56922C07K2319/00A61K2039/55544A61K2039/542G01N2469/20
Inventor 杜方川童文德朱白梅童武学谭稀
Owner CHENGDU OLYMVAX BIOPHARM
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