Preparation method and application of recombinant lactococcus lactis

A technology of Lactococcus lactis and lactic acid bacteria, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as heavy workload, complicated separation and purification process, and complex components of inactivated vaccines, so as to improve infection rate. The effect of the ability

Active Publication Date: 2018-02-02
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the relatively complex components of inactivated vaccines, large differences in immune efficiency for different individuals, and relatively weak protective effects against intracellular parasitic bacteria, the virulence of attenuated vaccines may recover and the storage and transportation conditions are high, and the separation and purification process of genetically engineered subunit vaccines is complicated. , easy to be degraded, the immune protection effect of DNA vaccine is poor, and the injection immunization method is widely used, the workload is heavy, and the operability of large-scale immunization is small. At present, there is no commercial Streptococcus agalactiae vaccine available

Method used

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  • Preparation method and application of recombinant lactococcus lactis
  • Preparation method and application of recombinant lactococcus lactis
  • Preparation method and application of recombinant lactococcus lactis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Cloning of embodiment 1 Streptococcus agalactiae Sip gene

[0050] According to the Sip gene sequence of Streptococcus agalactiae published by GenBank (CP0001141), design specific upstream and downstream primers SipF (5'-CGG GGTACC CCCAAGAAACAGATACGACG-3′) and SipR (5′CCC AAGCTT TTA GTTAAAGGATACGTGAA-3′), SipF has a KpnⅠ restriction site (shown underlined), SipR has a HindⅢ restriction site (shown underlined) and 6 His tag sequences (shown in italics), primers were provided by Guangzhou Ai Synthesized by Base Biotechnology Co., Ltd. PCR amplification was performed using the tilapia-derived Streptococcus agalactiae genomic DNA as a template, and the total volume of the amplification system was 25 μL: 0.5 μL (10 mmol / L) for each of the upstream and downstream primers SipF and SipR, 0.5 μL template DNA, PremixTaq TM 12.5 μL, 11 μL of double distilled water. The PCR reaction conditions were pre-denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 30s, 52°C for ...

Embodiment 2

[0051] Example 2 Preparation of Lactococcus lactis expressing Sip protein recombinantly

[0052] Inoculate the DH5α-pMD19T-Sip strain verified by sequencing into LB (Amp + , 100 μg / mL) liquid medium to expand culture, extract plasmid pMD19T-Sip, pMD19T-Sip plasmid, pNZ8148 plasmid and pNZ8124 plasmid were respectively digested with restriction endonucleases KpnⅠ and HindⅢ, and the digestion conditions were 37°C Incubate for 4h. The Sip gene fragment, pNZ8148 plasmid and pNZ8124 plasmid after digestion were recovered by gel, and T 4 DNA ligase was used to connect the Sip gene fragment and the pNZ8148 plasmid or pNZ8124 plasmid overnight at 16°C, and the ligated product was transformed into E.coli MC1061 competent cells to construct MC1061-pNZ8124-Sip and MC1061-pNZ8148-Sip recombinant Escherichia coli, and positive clones were detected by PCR After detection and identification by plasmid digestion, further sequencing verification was carried out. The restriction map of pNZ812...

example 3

[0054] Example 3 Sip protein induced expression and its induction condition optimization

[0055] Recombinant Lactococcus lactis NZ9000-pNZ8124-sip and NZ9000-pNZ8148-sip were inoculated into 5 mL of BHI liquid medium (Cm + , 10 μg / mL), cultured overnight at 30°C; the next day, the culture was expanded at a ratio of 1:50, and the expression was induced when the culture reached OD600≈0.4. The induction concentration optimization experiment of the inducer is to add nisin inducers with different concentration gradients (0ng / mL, 10ng / mL, 100ng / mL and 1000ng / mL), and induce at 30°C for 4h; the induction time optimization experiment is to use the best induction concentration Under the conditions, set the induction time gradient as 0h, 1h, 2h, 4h, 6h and 8h; after induction, centrifuge at 8000g for 10min to collect the bacteria, add an equal volume of PBS buffer according to the weight ratio of the bacteria at 1:1, and grind with liquid nitrogen Break up the cells, centrifuge to sav...

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Abstract

The invention relates to a preparation method and application of a recombinant lactococcus lactis. The invention is characterized in that the surface immunogenic protein (Sip) of tilapia-source streptococcus agalactiae is intracellularly or secretorily expressed in lactococcus lactis cells, vectors which are used for the recombinant expression of the Sip are pNZ8124 and pNZ8148 respectively, signal peptide segments are removed from Sip gene segments inserted into the vectors, and histidine sequence tags are added to the Sip gene segments, the recombinant protein is induced to be expressed by Nisin, the optimum induction condition is 4h of induction by 100ng / mL of Nisin, the optimum oral immunization concentration is 2.24*10<10>CFU / mL, and the oral dosage is 100 mu L. The recombinant lactococcus lactis disclosed by the invention which is applied in lactobacillus living vector vaccines against tilapia streptococcus agalactiae has the advantages of wide serotype coverage, direct oral administration, high safety, easiness in operation, easy large-scale herd immunity, good immune effect and the like.

Description

Technical field: [0001] The present invention relates to a preparation method and application of a tilapia source Streptococcus agalactiae lactic acid bacteria live carrier oral vaccine, specifically using Lactococcus lactis live cells as a carrier to prepare for preventing tilapia Streptococcus agalactiae (Streptococcus agalactiae, Oral vaccines for GBS) infection and methods of application thereof. Background technique: [0002] Tilapia (Tilapia) is a tropical fish native to Africa, belonging to Perciformes, Perciformes, Cichlidae, Tilapia (also known as Cichlidae, Cichlids belongs to). Because it has the characteristics of fast growth, miscellaneous food habits, strong fecundity, no intermuscular spines and rich nutrition, it is loved by farmers and consumers. Tilapia is widely farmed in Guangdong, Hainan, Guangxi, Fujian and other provinces in the south of my country. The scale and density of farming are increasing continuously, and it has become the most exported fres...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/31A61K39/09A61P31/04
CPCA61K39/092A61K2039/523A61K2039/542A61K2039/552C07K14/315C12N15/746
Inventor 刘志刚卢迈新蔡玉臻可小丽张德锋曹建萌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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