Construction method of specificity salmonella H:z29 diagnostic serum engineering strain

A technology of engineering strains and Salmonella, which is applied in the field of engineering strains and construction of specific Salmonella H:z29 diagnostic serum, can solve the problems of long time consumption, low specificity, and heavy workload, and achieve good feasibility and practicability Strong, to achieve effective removal effect

Active Publication Date: 2014-01-08
NANKAI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional method is time-consuming and labor-intensive
And the obtained i

Method used

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  • Construction method of specificity salmonella H:z29 diagnostic serum engineering strain
  • Construction method of specificity salmonella H:z29 diagnostic serum engineering strain
  • Construction method of specificity salmonella H:z29 diagnostic serum engineering strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construction of deletion strains

[0050] 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 .

[0051] The specific steps are as follows:

[0052] 1, f gene deletion

[0053] (1) Target strain:

[0054] The existing strain G4831 (6,8:z10:e,n,z15) in the laboratory;

[0055] (2) f Design of gene deletion primers:

[0056] 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

[0100] Surface flagellar antigen synthesis gene deletion strain G4831△ f △ wxya Analysis of the biological characteristics of

[0101] (1) Surface flagellar antigen synthesis gene deletion strain G4831△ f △ wxya Genetic Stability Analysis of

[0102] 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 with wxya The absence of is very stable (see for details Figure 4 ).

[0103] (2) Surface flagellar antigen synthesis gene deletion strain G4831△ f △ wxya Analysis of the growth characteristics of

[0104] Deletion strain G4831△ f △ wxya On the 0.3% semi-solid flat plate, it presents an obvious immobility state ...

Embodiment 3

[0106] Recombinant plasmid pTrc99a- f (z29) build

[0107] 1. flagellin antigen z29, f Gene, amplified from an existing laboratory strain G4890 (57:z29:z42).

[0108] 2. Due to f Both ends of the gene are highly conserved, download the related flagellar antigen z29 gene from ncbi, after comparison, design f Gene universal amplification primers.

[0109] 3. Cloning vector: plasmid pTrc99a, selected restriction site: Nco and BamH .

[0110] 4. PCR amplification of the target gene.

[0111] 1) Amplification primers: design Nco at both ends and BamH Restriction site, the amplification length is about 1.5kb;

[0112]Primer1: 5′-CATG CCATGG CACAAGTCATTAATACAAAC-3′ (Nco )

[0113] Primer2: 5′-CG GGATCC TTAACGCAGTAAAGAGAGGACGT-3′ (BamH )

[0114] 2) PCR system (50 μL):

[0115] 10× Pfu buffer with MgSO 4 (Thermo Scientific) 5 μL

[0116] MgSO 4 (Thermo Scientific) 2 μL

[0117] dNTP 4μL

[0118] genome (G4890) 1 μL

[0119] Primer1 1 μL

[0120...

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Abstract

The invention discloses a construction method of a specificity salmonella H:z29 diagnostic serum engineering strain. The construction method is characterized in that a modern molecular biological technology is used for transforming a conventional multi-clone antibody preparation system, and surface flagellar antigen synthetic gene deleted host bacteria are constructed; surface flagellar antigen synthetic genes of different pathogenic microorganisms are cloned to carriers suitable for expression, and the carriers are transferred to the antigen gene deleted host bacteria so as to obtain a series of engineering strains having the same genetic background and containing different pathogenic microorganism specificity surface flagellar antigen genes. The technology system can be used for constructing a special antibody library comprising a plurality of salmonellae. The antibody library can be used for supplementing existing anti-serum types and meanwhile can provide basis 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:z29 diagnostic serum and a construction method thereof. Background technique [0002] salmonella( Salmonella ) is a non-spore-forming, non-encapsulated Gram-negative bacillus. Except for Salmonella pullorum and Salmonella gallinarum typhi, the rest have flagella and can move. Most have pili, which can be adsorbed on the cell surface. Salmonella is the most important pathogenic genus in the Enterobacteriaceae family. About 2500 serotypes have been found in the world, and all of these serotypes can cause foodborne diseases. Salmonella is an important pathogen causing food poisoning. Among all kinds of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella ofte...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12R1/42
Inventor 冯露蒋新蕾王磊
Owner NANKAI UNIV
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