Application of Mycoplasma hyorhina Outer Membrane Protein mhr_0493 in the Preparation of Mycoplasma hyorhina or Its Antibody Detection Kit
A technology of Mycoplasma hyorrhinos, outer membrane protein, which is applied in measurement devices, instruments, peptides, etc., can solve the problems of diagnosis kits without Mycoplasma hyorrhinos, little knowledge of infection, pathogenicity and immunological status, and lag in research on Mycoplasma hyorrhinos. , to achieve the effect of easy purification and preparation, lower production cost, and improved specificity
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Embodiment 1
[0025] Screening of specific genes of Mycoplasma hyorhinosum
[0026] 1. Mycoplasma whole genome sequence information
[0027] The genome sequences of mycoplasma involved in this study were all downloaded from GeneBank, and the specific strain information of mycoplasma is shown in (Table 1).
[0028] Table 1 Mycoplasma bacterial strain information used in this application
[0029]
[0030]
[0031] 2. Analysis of specific genes of Mycoplasma hyorhinosum
[0032] Taking the Mycoplasma hyorhini genome as the research object, using BLAST and tBLASTn programs, all the genes on the genome were compared to other Mycoplasma genomes to obtain a high-scoring pair (HSP) group. In order to obtain more accurate data, we extracted HSPs (E-value=1e-4) whose similarity and sequence coverage were both greater than 80%, and further determined HSPs by two-way comparison. The analysis of homologous genes is a very complicated process, and there are many different algorithms and database...
Embodiment 2
[0036] Prokaryotic expression and purification of Mhr_0493 protein:
[0037] Construction of A.pET30a-Mhr_0493 fusion gene
[0038] 1. Synthesis of Mhr_0493 gene
[0039]Mhr_0493 (GenBank accession number ADM21950. 1) was used as a template, its transmembrane region was deleted, and one UGA codon was adjusted to UGG according to the codon preference of Escherichia coli to obtain the artificially synthesized Mhr_0493 outer membrane gene of Mycoplasma hyorhini (shown in SEQ ID NO.1).
[0040] 2. Construction of pET30a-Mhr_0493 recombinant plasmid
[0041] The artificially synthesized Mhr_0493 gene was cloned into the corresponding site (BamH I and Xho I) of the prokaryotic expression vector pET-32a (+) (Novagen, Germany) by BamH I and Xho I to obtain the recombinant plasmid pET30a-Mhr_0493 expressing the Mhr_0493 gene , It was confirmed by enzyme digestion and PCR that the plasmid was constructed correctly, and no base mismatch was confirmed by sequencing. The structural dia...
Embodiment 3
[0067] Antigen reactivity verification of Mycoplasma hyorhinois Mhr_0493
[0068] The purified rMhr_0493 protein was added with 5×Loading Buffer, boiled in boiling water at 100° C. for 10 minutes, cooled on ice for 10 minutes, and subjected to 12% SDS-PAGE electrophoresis. After electrophoresis, use nitrocellulose membrane to transfer protein, use 5% skimmed milk after transfer, block overnight at 4°C, wash membrane three times with TBST, each time for 5 minutes, and then incubate with artificially infected Mycoplasma hyorhini positive serum for 1 hour (Positive serum was diluted with TBST at a volume ratio of 1:1000), and a negative serum control group was set up at the same time. The membrane was washed three times with TBST for 5 minutes each time, and then incubated with HRP-labeled goat anti-pig enzyme-labeled secondary antibody for 1 hour (volume ratio 1:3000). After the incubation was complete, the membrane was washed five times with TBST for 5 min each, and developed ...
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