Construction method of mannheimia haemolytica LKTA protein prokaryotic expression vector 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 that there is no immunological method for detecting hemolytic mansoni bacillus, and there is no antigenic marker that can effectively detect hemolytic bacilli. Fast and accurate results
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Embodiment 1
[0092] 1. Target fragment lkta amplification
[0093] 1.1 Primers
[0094] Primer sequence lkta-F: CGC CATATG GATGGTGCAGCAAGTTC
lkta-R: CCG CTCGAG AGCAAAAATCAGCCTCTC
[0095] Underlined are restriction sites NdeI and XhoI
[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 ddH2O)
[0099]
[0100] 1.3 PCR program
[0101]
[0102]
[0103] 1.4 After the completion of electrophoresis, such as figure 1 ;
[0104] 1.5 Recover the purified fragment for enzyme digestion;
[0105] The PCR purification kit used was produced by Sangon Bioengineering (Shanghai) Co., Ltd.
[0106] 2. Enzyme digestion of target fragment and vector
[0107] 2.1 Digest the PCR product above
[0108] 1), 50 μl of enzyme digestion system for PCR product fragments
[0109]
[0110] Put the above system into a ...
Embodiment 2
[0127] Induced Expression and Purification of LKTA Recombinant Protein
[0128] Select the best induction condition for small test culture
[0129] ◆Pick a single colony of the expression strain Rosetta (DE3) in a test tube containing 2.5 mL of LB medium (30 μg / mL kanamycin and 34 μg / mL chloramphenicol), and cultivate overnight at 37° C. on a shaker at 220 rpm. LB Borth is provided by Sanko. When used, 25g of powder is dissolved in 1L dd H2O, and sterilized by high temperature and high pressure steam.
[0130] ◆Inoculate the cultured bacterial solution into 2.5mL LB medium at a volume ratio of 1:100, add 30μg / mL kanamycin and 34μg / mL chloramphenicol to the LB medium, 37°C, 220rpm Cultivate for about 3 hours.
[0131] ◆When the OD value reaches 0.6, add IPTG with a final concentration of 0.5mM, 220rpm, induce overnight at 20°C; induce 4h at 37°C, and use no IPTG inducer as a negative control.
[0132] ◆Centrifuge at 4000rpm for 10min to collect the bacteria, discard the supe...
Embodiment 3
[0172] Experimental program
[0173] 1 material
[0174] 1.1 Antigen
[0175] Indirect ELISA antigen A1 serotype LKTA recombinant protein was expressed and purified by our research group. The purified antigen concentration was 1 mg / mL.
[0176] 1.2 Animal serum
[0177] 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.
[0178] 1.3 Other reagents
[0179] 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.
[0180] 2 methods
[0181] Dilute the ELISA recombinant antigen to 50ng / ml with 0.05mol / L, pH9.6 carbonate buffer solution (CBS), coat the reaction plate, 100μL per well, and put it in a ...
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