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Building method of mannheimia haemolytica PlpE protein prokaryotic expression carrier and kit for detecting mannheimia haemolytica

A technology of mansoni bacillus and construction method, which is applied in the field of genetic engineering, can solve the problems such as the lack of immunoassay detection of hemolytic mansoni bacillus, and achieve the effect of fast detection speed and accurate results

Pending Publication Date: 2019-03-26
INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is no antigenic marker and preparation for effective detection of Mandella hemolyticus. Therefore, there is no immunological detection method in the art to detect Mandella hemolyticus

Method used

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  • Building method of mannheimia haemolytica PlpE protein prokaryotic expression carrier and kit for detecting mannheimia haemolytica
  • Building method of mannheimia haemolytica PlpE protein prokaryotic expression carrier and kit for detecting mannheimia haemolytica
  • Building method of mannheimia haemolytica PlpE protein prokaryotic expression carrier and kit for detecting mannheimia haemolytica

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] 1. Target fragment plpE amplification

[0093] 1.1 Primers

[0094] Primer

sequence

plp e-F

cgc catatg ggaggaagcggtagcg (SEQ ID No. 1)

plpe-R

ccg ctcgag ttattttttctcgctaac (SEQ ID No. 2)

[0095] The restriction sites NdeI and XhoI are underlined

[0096] 1.2 Amplification system

[0097] The PCR reaction uses pfu high temperature polymerase.

[0098] The amount of each component of PCR: (primer concentration is 1OD dissolved in 400μl ddH 2 O).

[0099] Upstream primer plpE-F

2μl

Downstream primer plpE-R

2μl

bacterial total DNA

2μl

dNTP

1μl (25mMeach)

10X pfu Buffer

5μl

Pfu

0.4μl (5μ / μl)

wxya 2 o

Make up to 50μl

[0100] 1.3 PCR program:

[0101]

[0102] 1.4 After the completion of electrophoresis, such as figure 1 . The target band is 1023bp.

[0103] 1.5 Recover the purified fragments for enzyme digestion

[0104] The PCR pur...

Embodiment 2

[0129] 2 small test culture selection of the best induction conditions

[0130] ◆Pick a single colony of expressing strain BL21(DE3) in a test tube (4mL LB medium, LB Borth provided by Sanko, Cat. No. A507002-0250, use 25g with 1L ddH 2 O dissolved. 30 μg / mL kanamycin) overnight at 37°C and 220 rpm.

[0131] ◆Inoculate the cultured bacteria into 4mL LB medium at a ratio of 1:100, add 30μg / mL kanamycin, and culture at 37°C and 220rpm.

[0132] ◆When the OD value reaches about 0.6, add IPTG with a final concentration of 0.5mM, 220rpm, and induce overnight at 20°C; induce for 4 hours at 37°C, and use no IPTG inducer as a negative control.

[0133] ◆Centrifuge at 4000rpm for 10min to collect the bacteria, discard the supernatant, suspend the bacteria with 500μL PBS (PH7.4) buffer, ultrasonically break for 6min, stop for 0.5s for 1.5s, centrifuge to collect the supernatant and precipitate, and dissolve the precipitate with 500μL inclusion body Solution (8Murea, 50mM Tris-HCl, 15...

Embodiment 3

[0181] An indirect ELISA method for the detection of Mannella hemolytica using PLPE protein as the coated antigen.

[0182] 2. Experimental plan

[0183] 1 material

[0184] 1.1 Antigen

[0185] Indirect ELISA antigen PLPE recombinant protein was expressed and purified by our research group. The purified antigen concentration was about 1mg / mL.

[0186] 1.2 Animal serum

[0187] Three kinds of goat-positive sera, including the standard positive serum of Mannella hemolyticus rats, standard negative serum, Escherichia coli, streptococcus, and Mycoplasma ovis pneumoniae, were all preserved by our research group.

[0188] 1.3 Other reagents

[0189] HRP-goat anti-mouse IgG was purchased from Huamei Bioengineering Company, tetramethylbenzidine (TMB) was purchased from Tiangen Biochemical Technology Co., Ltd., and 96-well strips for ELISA test were purchased from Bao Bioengineering (Dalian) Co., Ltd.

[0190] 2 methods

[0191] Dilute the ELISA recombinant antigen to a certain m...

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Abstract

The invention provides a building method of a mannheimia haemolytica PlpE protein prokaryotic expression carrier and a kit for detecting mannheimia haemolytica, and belongs to the field of genetic engineering. The mannheimia haemolytica PlpE protein is built in the prokaryotic expression carrier for the first time; the prokaryotic expression carrier of the mannheimia haemolytica PlpE protein is obtained; through recombinant expression, antigen marker-PlpE protein for detecting the mannheimia haemolytica is obtained; the expressed PlpE is subjected to Western Blot analysis to obtain a 47KDa target strip band. Experiments show that the PlpE protein is used as antigen to be subjected to immunoreaction with three kinds of bacterial positive serum such as escherichia coli, streptococcocci and mycoplasma pneumoniae; the result shows that the result is negative. The mannheimia haemolytica PlpE protein provided by the invention has high specificity and can be used as an antigen marker for detecting the mannheimia haemolytica.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a prokaryotic expression vector of the Mansonella hemolyticus PlpE protein and a kit for detecting the Mansonella hemolyticus. Background technique [0002] Mannella hemolyticus, formerly known as Pasteurella hemolyticus, is a Gram-negative, immobile, non-spore-forming, oxidase-positive, Swiss stain bipolar facultative anaerobic bacillus. On blood agar, freshly isolated colonies were weakly beta-hemolyzed. It is further divided into two distinct biotypes (A, T) based on the ability to ferment arabinose and trehalose. Twelve A biotypes (serotypes 1, 2, 5, 6, 7, 8, 9, 12, 13, 14, 16, 17) and four T biotypes (serotypes 3, 4, 10, 15) has been identified. Mannella hemolyticus is a zoonotic disease that seriously endangers the livestock breeding industry, and can cause serious diseases such as hemorrhagic sepsis in cattle and sheep c...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/70C07K14/195G01N33/569
CPCC07K14/195C12N15/66C12N15/70G01N33/56911
Inventor 张月梅赵世华宋越戴伶俐王娜刘威张帆杨斌达来宝力格陈伟
Owner INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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