Construction method for engineered strain capable of preparing series of specific salmonella diagnostic serum

A technology of engineering strains and Salmonella, which is applied in the field of engineering strains and construction of specific Salmonella H:k diagnostic serum, can solve the problems of time-consuming, heavy workload, and low specificity, and achieve good feasibility and strong practicability , the practical effect of the method

Inactive Publication Date: 2014-01-01
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional method is time-consuming and labor-intensive
And the obtained is a polyclonal serum, its specificity is not high, and some cross-reactions often occur

Method used

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  • Construction method for engineered strain capable of preparing series of specific salmonella diagnostic serum
  • Construction method for engineered strain capable of preparing series of specific salmonella diagnostic serum
  • Construction method for engineered strain capable of preparing series of specific salmonella diagnostic serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction of deletion strains

[0051] First transform the pSim plasmid into the target strain (G4831), and then use the pSim bacterial DNA recombination system to f The gene was replaced with the chloramphenicol acetyltransferase gene on the plasmid pKD3, and the wxya The gene was replaced with the kanamycin resistance gene on the plasmid pKD4, and the deletion strain was initially screened out by antibiotics, and then identified by PCR and sequencing, and finally the corresponding deletion strain G4831△ f △ wxya .

[0052] The specific steps are as follows:

[0053] 1, f gene deletion

[0054] (1) Target strain:

[0055] The existing strain G4831 (6,8:z10:e,n,z15) in the laboratory;

[0056] (2) f Design of gene deletion primers:

[0057] for f Gene (about 1.5kb) deletion primers use the chloramphenicol acetyltransferase gene on pKD3 as a PCR template, and add corresponding f The homologous sequence of the upstream and downstream of the gene is a...

Embodiment 2

[0101] Surface flagellar antigen synthesis gene deletion strain G4831△ f △ wxya Analysis of the biological characteristics of

[0102] (1) Surface flagellar antigen synthesis gene deletion strain G4831△ f △ wxya Genetic Stability Analysis of

[0103] The deletion strain was continuously passed on LB solid medium for 10 generations, and each generation was used f , wxya The primers were identified to identify the genetic stability, and the results showed that only fragments of 1.1kb and 518bp could be amplified for 10 consecutive generations. Explain that the Salmonella deletion strain G4831△ prepared by the present invention f △ wxya of f and wxya The absence of is very stable (see for details Figure 4 ).

[0104] (2) Surface flagellar antigen synthesis gene deletion strain G4831△ f △ wxya Analysis of the growth characteristics of

[0105] Deletion strain G4831△ f △ wxya On the 0.3% semi-solid flat plate, it presents an obvious immobility state (...

Embodiment 3

[0107] Recombinant plasmid pTrc99a- f (k) construction

[0108] 1. flagellin antigen K, f Gene, amplified from the existing strain G4816 (6,7:k:1,5) in the laboratory.

[0109] 2. Due to f Both ends of the gene are highly conserved, download the related flagellar antigen k gene from ncbi, after comparison, design f Gene universal amplification primers.

[0110] 3. Cloning vector: plasmid pTrc99a, selected restriction site: Nco and BamH .

[0111] 4. PCR amplification of the target gene.

[0112] 1) Amplification primers: design Nco at both ends and BamH Restriction site, the amplification length is about 1.5kb;

[0113] Primer1: 5′-CATG CCATGG CACAAGTCATTAATACAAAC-3′ (Nco )

[0114] Primer2: 5′-CG GGATCC TTAACGCAGTAAAGAGAGGACGT-3′ (BamH )

[0115] 2) PCR system (50 μL):

[0116] 10× Pfu buffer with MgSO 4 (Thermo Scientific) 5 μL

[0117] MgSO 4 (Thermo Scientific) 2 μL

[0118] dNTP 4μL

[0119] genome (G4816) 1 μL

[0120] Primer1 1 μL ...

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Abstract

The invention discloses a construction method for an engineered strain capable of preparing a series of specific salmonella diagnostic serum. The method utilizes a modern molecular biological technology to modify a traditional polyclonal antibody preparation system so as to construct host bacteria lack of surface flagellar antigen synthesis genes; the surface flagellar antigen synthesis genes of different pathogene microbes are cloned on suitable expression vectors, and then the vectors are transferred to the host bacteria lack of the antigen genes, so as to obtain a series of engineered strains which have the same genetic background, and contain specific surface flagellar antigen genes of the different pathogene microbes. Due to the adoption of the technologies, a library containing a plurality of specific salmonella antibodies can be constructed. The antibody library can supplement current antiserum types, and meanwhile can lay foundation for the application of other immunology technologies.

Description

technical field [0001] The invention belongs to the technical field of biological products, and relates to an engineering strain capable of preparing specific Salmonella series diagnostic serum, more specifically an engineering strain capable of preparing specific Salmonella H:k diagnostic serum and a construction method thereof. Background technique [0002] salmonella( Salmonella ) belongs to Enterobacteriaceae ( Enterobacteriaceae ), which are Gram-negative bacteria. Some Salmonella are pathogenic to humans, some are only pathogenic to animals, and some are pathogenic to both humans and animals. Salmonellosis is caused by Salmonella spp. Salmonella ) is a general term for various diseases caused by different serotypes infecting various animals. People infected with Salmonella or the feces of carriers can contaminate food and cause food poisoning. [0003] Flagella is an accessory structure on the surface of Gram-negative bacteria that is responsible for bacterial mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/42
Inventor 冯露蒋新蕾王磊
Owner NANKAI UNIV
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