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Storage method of transgenic streptococcus suis serotype MRP engineered microorganisms

A technology of genetically engineered bacteria and preservation methods, which is applied in the field of preservation of Streptococcus suis type 2 MRP genetically engineered bacteria, can solve the problems of high equipment requirements, easy pollution, and frequent passage, so as to improve production efficiency and quality, reduce The effect of passage times and reduced storage space

Inactive Publication Date: 2018-04-06
HANBANG MEDICAL SCI & TECH HARBIN CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the vacuum cryopreservation method has the best effect, but it requires high equipment, high cost and complicated preparation process.
The low-temperature storage method on agar slant is easy to operate, easy to popularize, and has a wide range of applications. The disadvantage is that it needs to be frequently subcultured, and it is prone to pollution. The storage period is short, and it is not suitable for bacteria with high nutritional requirements.
Defiberized sheep blood cryopreservation method, the sheep blood used in this method is not easy to obtain for general laboratories, and cannot be widely used
Liquid paraffin preservation method has poor preservation effect on bacteria

Method used

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  • Storage method of transgenic streptococcus suis serotype MRP engineered microorganisms
  • Storage method of transgenic streptococcus suis serotype MRP engineered microorganisms
  • Storage method of transgenic streptococcus suis serotype MRP engineered microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preservation method of the Streptococcus suis type 2 MRP genetically engineered bacteria provided in this example is operated as follows.

[0044] 1 Obtain the engineering bacteria liquid containing the MRP gene sequence of Streptococcus suis type 2

[0045] Get the bacterium solution of the transgenic Streptococcus suis type 2 partial gene Escherichia coli (target bacterium) transformed with the Streptococcus suis type 2 partial gene sequence and inoculate it into 200mL of LB broth medium (containing 100mg / L ampicillin), 37°C, 180rpm / min shaking culture for 4h.

[0046] 2 Take the cultured bacteria solution and mix it with the preservation reagent at a volume ratio of 1:1 to obtain the target bacteria preservation solution, and then divide it into sterilized 1.5mL EP tubes (each EP tube contains 1mL target bacteria preservation solution ) and quickly stored at -20°C.

[0047] Wherein, the preservation reagent used is physiological saline containing glycerol, the...

experiment example 1

[0052] The transgenic Escherichia coli were taken out at different time points (6 months, 9 months, 12 months, 18 months, 24 months) after storage for stability investigation. details as follows.

[0053] 1 strain survival rate

[0054] At different time points, the Streptococcus suis type 2 MRP gene Escherichia coli preserved by the storage method in Example 1 was taken out from the -20°C environment, and 30 tubes were taken each time, and quickly thawed in a 37°C water bath, and then streaked on the plate. For the recovery of bacterial strains, one tube is streaked on one plate, and a total of 30 plates are streaked. Observe whether there are colonies growing on each plate. If there are colonies, it is recorded as survival. If no colonies grow, it is recorded as death. The survival rate (number of surviving plates / total number of streaked plates) of the bacterial strain after recovery after storage for different periods of time is shown in Table 1 below.

Embodiment 2

[0079] The preservation method of the Streptococcus suis type 2 MRP genetically engineered bacteria provided in this example is operated as follows.

[0080] 1 Obtain the engineering bacteria liquid containing the MRP gene sequence of Streptococcus suis type 2

[0081] Get the bacterium solution of the transgenic Streptococcus suis type 2 partial gene Escherichia coli (target bacterium) transformed with Streptococcus suis type 2 partial gene sequence and inoculate in 200mL LB broth medium (containing 100mg / L ampicillin), 37°C, 200rpm / min shaking culture for 4h.

[0082] 2 Take the cultured bacteria solution and mix it with the preservation reagent at a volume ratio of 1:1 to obtain the target bacteria preservation solution, and then distribute it into sterilized 1.5mL EP tubes (each EP tube contains 1mL target bacteria preservation solution ) and quickly stored at -80°C.

[0083] Wherein, the preservation reagent used is physiological saline containing glycerol, the concent...

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Abstract

The invention discloses a preservation method for transgenic streptococcus suis type 2 MRP genetically engineered bacteria, and relates to the technical field of transgenic engineering. The preservation method of Streptococcus suis type 2 MRP genetically engineered bacteria disclosed in the present invention takes the physiological saline containing glycerin as the preservation reagent, adds the preservation reagent into the bacterial liquid containing the target bacteria, and distributes it in sterilized EP tubes , and immediately placed at -80°C to -20°C for low-temperature storage; this storage method is simple to operate, has a long storage time, takes up a small space, and is low in cost. It can be stored in multiple tubes in small quantities at one time, which is convenient for management and prevents repetition Freezing and thawing causes bacterial death and bacterial contamination, improving the survival rate of bacterial cells; and after long-term storage, it still maintains the same vitality and purity as the original strains, which plays an important role in maintaining the stability of bacterial strains and plasmids. effect.

Description

technical field [0001] The invention relates to the technical field of transgenic engineering, in particular to a preservation method for transgenic Streptococcus suis type 2 MRP genetically engineered bacteria. Background technique [0002] Streptococcus suis is a Gram-positive coccus with an capsule. It can be roughly classified as Lancefield group D (Lancefieldgroup D) group D streptococcus according to its cell wall antigenic composition. According to the different capsular antigens (CPS), Streptococcus suis is divided into 35 (types 1-34, type 1 / 2) serotypes, of which types 1, 2, 7, and 9 are pathogenic bacteria of pigs. The colonization site of Streptococcus suis is the upper respiratory tract of pigs, especially the tonsils and nasal cavity. Some serotypes of Streptococcus suis are pathogenic and mainly infected through wounds. It can cause acute sepsis (septicemia with sudden death), meningitis (meningitis), arthritis (arthritis), endocarditis (endocarditis), pneu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04C12N1/21C12R1/19
CPCC12N1/04C07K14/315C12N1/20
Inventor 余美伦李影秦香芹孟庆雪韩伟丽何佳苑玉凤王宁王春东闫增福刘庆宇
Owner HANBANG MEDICAL SCI & TECH HARBIN CITY
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