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Recombinant lactococcus lactis and tilapia streptococcus agalactiae disease vaccine

A technology of Lactococcus lactis and Streptococcus lactis, applied in the direction of recombinant DNA technology, bacteria, antibacterial drugs, etc., can solve the problems of chemical drug resistance, drug residue quality and safety issues, etc.

Active Publication Date: 2020-01-10
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

Chemical drug prevention and control is the most direct, convenient and convenient means of prevention and control, but long-term use of chemical drugs is prone to drug resistance, resulting in quality and safety problems caused by drug residues

Method used

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  • Recombinant lactococcus lactis and tilapia streptococcus agalactiae disease vaccine
  • Recombinant lactococcus lactis and tilapia streptococcus agalactiae disease vaccine
  • Recombinant lactococcus lactis and tilapia streptococcus agalactiae disease vaccine

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Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: Construction carries the recombinant plasmid of sip-pgk fusion gene

[0026] The sip gene and the pgk gene were amplified from Streptococcus agalactiae (WC1535) preserved by the applicant, and the pNZ8148 plasmid was used as a carrier plasmid to construct a recombinant plasmid carrying the sip-pgk fusion gene.

[0027] The PCR primer sequences for amplifying the sip gene and the pgk gene are shown in Table 1 below, in which a 6×His tag is added to the end of the primer for the target gene, and the general primer of pNZ8148 is selected as the identification primer. The primers used were synthesized at Guangzhou Aiji Biotechnology Co., Ltd. The sequence marked in italics is the 15 bp base sequence homologous to the pNZ8148 vector plasmid, and the sequence marked underlined is the base sequence corresponding to the 6×His tag.

[0028] Table 1

[0029]

[0030] Streptococcus agalactiae WC1535 was cultured in BHI medium (purchased from Guangzhou Kanglong ...

Embodiment 2

[0036] Example 2: Construction of recombinant Lactococcus lactis and identification of fusion gene expression

[0037] First, Lactococcus lactis (Lactococcus. lactis) NZ9000 (L. lactis NZ9000) (purchased from REBIO) competent cells were prepared. A single colony of Lactococcus lactis NZ9000 was cultured statically at 30° C. for 6 h in M17 liquid medium (purchased from Guangzhou Kanglong Biotechnology Co., Ltd.) containing 0.5% glucose. Take the above culture in the M17 liquid medium containing 0.5% glucose + 1% glycine at a ratio of 1:10, and culture overnight at 30°C. According to the ratio of 1:10, take the above culture in the solution containing 0.5% glucose + 0.5mol·L -1 Sucrose + 2% glycine M17 liquid medium, continue static culture to OD 600 = 0.5. Centrifuge the culture at 5000g for 15min at 4°C, discard the supernatant, and add 1 volume of pre-cooled solution (0.5mol·L -1 sucrose+10% glycerin) to resuspend, centrifuge at 5000g at 4°C for 15min, discard the super...

Embodiment 3

[0043] Embodiment 3: the immunoprotective effect of recombinant Lactococcus lactis

[0044] 420 healthy tilapias were selected from Guangdong Tilapia Breeding Farm, with a body length of 8±1cm and a weight of 14±1g.

[0045] These tilapias were randomly divided into 12 groups, 35 fish in each group, and the immune experiment was carried out after the tilapias were kept stable for a while. The 12 groups were divided into 2 large groups, 6 groups received the second immunization 7 days after the first immunization, that is, the second immunization (day 14), and the remaining 6 groups received 7 days after the second immunization (14 days). Carry out the third immunization, that is, three immunizations (day 21), and the immunization method is immunization every other week.

[0046] The six experimental groups were L.lactis NZ9000pNZ8148-sip-pgk (Sip-Pgk fusion protein lactic acid bacteria vaccine), L.lactis NZ9000pNZ8148-sip (Sip protein lactic acid bacteria vaccine control), ...

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Abstract

The invention relates to recombinant Lactococcus lactis comprising a fusion gene of a sip gene and a pgk gene of Streptococcus agalactiae, and the fusion gene is introduced into the Lactococcus lactisby a plasmid vector. The fusion gene has a DNA sequence as shown in SEQ No. 1. The plasmid vector is a pNZ8148 vector. The lactococcus lactis is Lactococcus. lactis NZ9000. The invention further provides a tilapia mossambica streptococcus agalactiae disease vaccine. The tilapia mossambica streptococcus agalactiae disease vaccine comprises the recombinant lactococcus lactis. Wherein the recombinant lactococcus lactis is the recombinant lactococcus lactis induced by nisin. The vaccine is the vaccine administrated by oral administration and intragastric administration. The tilapia mossambica streptococcus agalactiae disease vaccine comprises the recombinant lactococcus lactis with the concentration of 2*10 <10> cfu mL <-1>, and the administration dosage is 200 [mu]L.

Description

technical field [0001] The invention belongs to the technical field of prevention and control of aquatic organism diseases, and in particular relates to a recombinant Lactococcus lactis and a tilapia agalactiae streptococcosis vaccine comprising the recombinant Lactococcus lactis. Background technique [0002] Tilapia (Oreochromis sp.) is one of the main cultured fishes in southern my country. It has the advantages of miscellaneous food habits, wide adaptability, strong reproductive ability, and fast growth rate. In recent years, the outbreak of tilapia disease has caused huge losses to the tilapia industry, and the trend has become more serious, hindering the healthy development of the tilapia industry. The main pathogens of tilapia diseases are Streptococcus iniae and Streptococcus agalactiae, and more than 90% of the clinical strains are Streptococcus agalactiae. [0003] The main causes of tilapia streptococcosis agalactiae are poor water quality, high breeding density,...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/62A61K39/09A61P31/04C12R1/01
CPCC12N9/1217C07K14/315A61K39/09A61P31/04C12Y207/02003A61K2039/523Y02A40/81
Inventor 赖迎迢石存斌陶家发袁玉梅孙承文巩华
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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