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Preparation method and screening method of salmonella PagC protein monoclonal antibody

A technology of monoclonal antibodies and screening methods, applied in the direction of antibacterial immunoglobulin, immunoglobulin, chemical instruments and methods, etc., can solve serious cross-reaction of Enterobacteriaceae bacteria, insufficient sensitivity of serum samples, and complex antigenic epitopes And other issues

Active Publication Date: 2021-03-09
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

my country has listed the purification of Salmonella in the breeding environment as a key development project. The premise of the purification work first needs the support of precise detection methods. Now the widely used detection method of Salmonella in clinical practice is mainly the slide agglutination test, which is easy to operate Fast and low cost, but the detection throughput is low and the sensitivity to serum samples with low antibody titers is insufficient, and false negatives are prone to occur. The results are highly subjective and there are problems of cross-reaction with other Enterobacteriaceae bacteria Therefore, it is difficult for this method to provide strong support for the purification of salmonellosis in my country, and it is urgent to develop a new detection method to replace this traditional detection method
[0003] ELISA (Enzyme-Linked ImmunoSorbent Assay, ELISA) has been well known as a high-throughput, highly sensitive serological detection method. Establishing an ELISA detection method for Salmonella antibodies can effectively improve the efficiency and accuracy of clinical detection of Salmonella, but due to the serious cross-reaction of Enterobacteriaceae bacteria, it is necessary to overcome this problem first in the process of establishing the ELISA method
The detection antigens selected by the existing Salmonella antibody ELISA detection method are mainly O antigen and H antigen, but O and H antigen epitopes are complex, and there is still cross-reactivity when used in ELISA antibody detection

Method used

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  • Preparation method and screening method of salmonella PagC protein monoclonal antibody
  • Preparation method and screening method of salmonella PagC protein monoclonal antibody
  • Preparation method and screening method of salmonella PagC protein monoclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0035] see Figure 1-2 , the present invention provides a technical solution: preparation of antigen,

[0036] 1.1 Expression, purification and renaturation of PagC protein

[0037] The Salmonella PagC recombinant expression strain pET-28a-PagC was cultured to the logarithmic phase with LB (adding kanamycin with a final concentration of 50 ng / mL); Lactoside, Isopropylβ-D-Thiogalactoside) inducer, 37 ℃ for 3 hours of shaking culture; the induced cells were washed three times with sterilized PBS, and finally resuspended in PBS, then ultrasonically disrupted the cells (power 200W, work 5s interval 8s); 1 hour later, centrifuge at 4°C for 30 minutes, take the supernatant and precipitate for SDS-PAGE, determine the expression form of the protein, and then perform a large amount of expression, and purify according to the purification process of the inclusion body protein. It is confirmed that the purified protein needs to be further refolded. This time, the gradient dialysis method ...

Embodiment 2

[0043] see Figure 3-5 , the present invention provides a technical solution: the preparation of PagC protein monoclonal antibody,

[0044] 2.1 Animal immunity

[0045] Select SPF grade female Balb / c mice aged 6-8 weeks, and inject 50ug / 0.3mL subcutaneously on the back after emulsification with purified protein PagC for initial vaccination; 21 days later, inject 50ug / 0.5mL subcutaneously and intraperitoneally after antigen emulsification / mouse; 10 days after the second immunization, blood was collected to separate the serum, and the mouse titer was determined by the established indirect ELISA, and the mouse with the highest titer and greater than 104 was selected for intraperitoneal injection of 100ug / 0.5mL / antigen; 3 days later, splenocytes were collected for fusion . KLH-P2 immunized mice, the immunization dose was 100ug / 0.5mL / mouse, other methods refer to the above immunization method.

[0046] After the second immunization, the mouse titer reached 104, and splenocytes...

Embodiment 3

[0058] see Figure 6-7 , the present invention provides a technical solution: screening of specific monoclonal antibodies,

[0059] 3.1 Preliminary identification of B cell epitopes of Salmonella PagC protein monoclonal antibody by Dot-Blot

[0060] Add 3ug PagC, KLH-P1, KLH-P2, and KLH dropwise on the NC membrane, overnight at 4°C; add 5% skimmed milk to block at room temperature for 2h; wash with PBST 3 times, 5min / time; add positive hybridoma cell supernatant, Incubate at room temperature for 1 hour; wash with PBST 3 times, 5 minutes each time; add goat anti-mouse HRP enzyme-labeled secondary antibody diluted 1:5000, incubate at room temperature for 1 hour; wash with PBST 3 times, 5 minutes each time; develop color with DAB in the dark for 5-10 minutes, Rinse the NC membrane in distilled water to stop the color development, and take it out to dry.

[0061] The results showed that the J cell strain had an obvious reaction with KLH-P1, but had no reaction with KLH and KLH-P...

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Abstract

The invention discloses a preparation method and a screening method of a salmonella PagC protein monoclonal antibody. The preparation method comprises the following steps of immunizing Balb / c mice byusing purified PagC protein, and obtaining six hybridoma cells capable of producing an anti-PagC protein monoclonal antibody according to a hybridoma cell preparation technology; and synthesizing intergeneric specific amino acid sequences DRQASGSVEPEGIH(P1) and FKEHSTQDGDSFNKISSRKTGFA(P2) containing linear epitopes as screening antigens to obtain a monoclonal antibody cell strain J of which the antigen epitopes are on a linear P1 sequence, wherein the other five monoclonal antibody epitopes are not on linear P1 and P2 amino acid sequences. Western-blotting results show that mAb J can react with purified PagC protein, wild type pullorum disease and mouse typhoid fever PagC protein and does not react with other enterobacteriaceae PagC protein, and it is shown that the screened mAb J epitopeP1 is conservative in salmonella and is intergeneric specific. By preparing the monoclonal antibody, a salmonella antibody blocking ELISA detection method can be established, and the monoclonal antibody has the characteristics of good specificity and high sensitivity and has wide popularization potential.

Description

technical field [0001] The invention relates to the technical field of veterinary medicine, in particular to a preparation method and a screening method of a Salmonella PagC protein monoclonal antibody. Background technique [0002] Salmonella is a Gram-negative bacillus that parasitizes in the intestinal tract of humans and animals. It mainly causes diseases such as typhoid fever, paratyphoid fever, acute gastroenteritis, and sepsis in humans. Infecting animals can lead to sepsis, gastroenteritis, and local inflammation of other tissues. Seriously endanger public health and livestock and poultry breeding safety. my country has listed the purification of Salmonella in the breeding environment as a key development project. The premise of the purification work first needs the support of precise detection methods. Now the widely used detection method of Salmonella in clinical practice is mainly the slide agglutination test, which is easy to operate Fast and low cost, but the de...

Claims

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Application Information

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IPC IPC(8): C07K16/12G01N33/577G01N33/569
CPCC07K16/1235G01N33/577G01N33/56916G01N2333/255Y02A50/30
Inventor 马喆范红结郭晓
Owner NANJING AGRICULTURAL UNIVERSITY
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