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Recombinant bacterium for expressing SEF14 functional fimbriae and application of recombinant bacterium

A technology of recombinant strains and recombinant plasmids, which is applied in the fields of biomedicine and immunodiagnosis, and can solve the problems of lack of specific targeting

Active Publication Date: 2021-03-12
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the infection situation of Salmonella Enteritidis in chicken flocks is serious, which has brought a severe test to human food safety, but there is currently a lack of specific targeted detection and monitoring technology for Salmonella Enteritidis infection in chicken flocks to purify it

Method used

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  • Recombinant bacterium for expressing SEF14 functional fimbriae and application of recombinant bacterium
  • Recombinant bacterium for expressing SEF14 functional fimbriae and application of recombinant bacterium
  • Recombinant bacterium for expressing SEF14 functional fimbriae and application of recombinant bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Amplification and cloning of embodiment 1 Salmonella enteritidis sef14 operon

[0042] Using the genome of Salmonella enteritidis standard strain C7920 as a template, use P505 ultra-fidelity enzyme (purchased from Nanjing Novizan Biotechnology Co., Ltd.) to amplify the sef14 operon gene fragment, and its coding sequence without signal peptide is:

[0043]

[0044]

[0045]

[0046]

[0047] The upstream and downstream primers for amplifying the sequence are:

[0048] sef-up: 5'-G GCAT GC AAAATg gCg TgAgTATAT TAg CAT CCg CA-3';

[0049] set-lo: 5'-C G TCG AC TT ATT ATAATT CAA TTT CTG TCG CAT AT-3'.

[0050] Wherein the underline marks represent SphI and SalI restriction sites respectively, and the amplified product is 4268bp ( figure 1 ).

[0051] The sef14 operon gene fragment amplified by PCR and the pBR322 vector were digested with SphI and SalI, and then circularized in vitro by DNA ligase. The product was named pBR-sef14, and then transformed int...

Embodiment 2

[0052] Expression and identification of embodiment 2SEF14 pili and SefA recombinant protein

[0053] Expression of SEF14 pili: pBR-sef14 was transformed into Escherichia coli SE5000, cultured at 37°C for 12 hours, and pili were extracted by heat extraction method. SE5000 containing only pBR322 empty vector was used as negative control. The steps of the heat extraction method are: place the above bacterial solution at 4°C and centrifuge at 4500rpm for 10 minutes, discard the supernatant and wash twice with sterile PBS; then add 1M NaCl-0.05M Tris-HCl (pH=7.4) solution to resuspend the bacterial cells and Place in a water bath at 62°C for 30 minutes; centrifuge at 8,000 rpm for 20 minutes, transfer the supernatant to a new centrifuge tube for volume measurement, slowly add saturated ammonium sulfate dropwise to a final concentration of 25%, and precipitate overnight; centrifuge at 14,000 rpm for 30 minutes the next day, discard the supernatant, and use Resuspend the pellet in st...

Embodiment 3

[0060] Example 3 Indirect agglutination test of recombinant bacteria based on SEF14 pili to detect Salmonella Enteritidis infection in chicken flocks

[0061] After cultivating the recombinant bacteria expressing SEF14 pili obtained in Example 1-3 for 12 hours, the amount of bacteria was adjusted to be 5×10 9CFU / mL, then centrifuge at 4000rpm for 5min, discard the supernatant and wash twice with sterile PBS. Use PBS containing 0.25% formaldehyde to resuspend the bacteria, and place it overnight at 4°C as the agglutinating antigen. The above-mentioned agglutinated antigen was respectively mixed with 10 parts of SPF chicken infection Salmonella enteritidis serum (numbering is 1-10), 30 parts of clinical Salmonella enteritidis positive serum (numbering is 11-40), 10 parts of clinical Salmonella pullorum serum (numbering is 41-50) ), 10 clinical Salmonella typhi positive chicken serum (number 51-60), 10 clinical E. coli positive chicken serum (number 61-70), 10 SPF chicken infect...

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Abstract

The invention discloses a recombinant plasmid. The recombinant plasmid is obtained by inserting a sef14 operon gene into an expression vector pBR322. The invention also discloses a recombinant strain.The recombinant strain is obtained by introducing the recombinant plasmid into inert carrier bacteria S9. The invention also discloses a preparation method and an application of the recombinant strain. According to the invention, SEF14 is subjected to surface display on the inert carrier bacterium S9 for the first time to express single fimbriae SEF14, so that background non-specific reaction canbe avoided, salmonella enteritidis infection can be specifically detected at the same time, the method has the advantages of rapidness, specificity, sensitivity, simplicity, low cost and the like, and the requirements of on-site and large-scale detection can be met. The SEF14 functional bacterial hair can be used for detecting and monitoring salmonella enteritidis infected chicken flocks after single bacterial hair SEF14 is shown and expressed on the upper surface of an inert carrier, only 5-10 microliters of the detection sample and an isopyknic detection reagent are needed, the salmonella enteritidis infected chicken flocks can be detected and monitored by utilizing a simple glass plate agglutination reaction, and observing of a reaction result by naked eyes on site and accurately judging whether an animal is infected by salmonella enteritidis or not are carried out.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and immunodiagnosis, and specifically relates to a recombinant bacterium expressing SEF14 functional pili and its application. Background technique [0002] Salmonella enteritidis has no host specificity, is one of the main pathogenic bacteria that cause food poisoning in humans, and can cause non-typhoid gastroenteritis in humans, livestock and poultry. About 93.8 million people in the world are infected with Salmonella every year, and 155,000 people die; and in my country, Salmonella gastroenteritis caused by Salmonella accounts for about 40% of bacterial food poisoning every year, ranking first in bacterial food poisoning, and Salmonella Enteritidis It is the main cause of Salmonella food poisoning outbreaks. [0003] Livestock and poultry products are the main source of Salmonella Enteritidis infection, among which eggs are the most common carrier. This is because Salmonella Enteritidis ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/64C12N15/31C12N1/21C12Q1/10C12R1/42
CPCC07K14/255C12N15/74C12Q1/10
Inventor 侯千禧顾宣强朱国强刘家奇段强德夏芃芃
Owner YANGZHOU UNIV
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