Establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detection of Brucella abortus HSP70
A technology for establishing methods for Brucella bovis, which is applied in the field of establishing indirect ELISA, and can solve problems such as low isolation rate, high sensitivity of PCR detection method, false positives, etc.
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Embodiment 1
[0033] The PCR amplification of embodiment 1 HSP70 gene
[0034] The gene sequence of HSP70 was obtained from the genome of Brucella.abortus 2308. According to the expression plasmid pET32a and gene sequence analysis, BamHI and XhoI were selected as restriction sites, and Primer Premier 5.0 was used to design primers.
[0035] The primer sequences are as follows:
[0036] P1: 5'-GCggatccATGGCTAAAGTTATTGGTATC-3'; BamH I; SEQ ID NO: 1;
[0037] P2: 5'-GCctcgagTTAGCGGATCTGGTGCTCCTT-3'; Xho I; SEQ ID NO:2.
[0038] Using the genomic DNA of Brucella.abortus 2308 as a template, use the above primers for PCR amplification. The PCR reaction system is:
[0039]
[0040] The PCR reaction program was 94°C for 5min; 94°C for 30s, 56°C for 30s, 72°C for 1.5min, a total of 30 cycles; and finally 72°C for 5min.
[0041] After the reaction was completed, agarose gel electrophoresis was carried out, and the PCR product was recovered with a gel recovery kit to obtain a band with a size of...
Embodiment 2
[0042] Embodiment 2 Construction of recombinant expression vector and expression strain
[0043] The HSP70 gene obtained in Example 1 and the plasmid pET32a were subjected to double enzyme digestion, recovered from the gel, ligated and transformed, coated with LB / Amp plates, and cultured upside down in an incubator. Pick the single clone on the LB / Amp plate, shake the bacteria, and use the universal primers to carry out bacterial liquid PCR identification, and obtain the positive clone pET32a-HSP70;
[0044] Among them, the general primer sequence is as follows:
[0045] S-Tag primer: 5'-CGAACGCCAGCACATGGACA-3'; SEQ ID NO: 3;
[0046] T7 terminatorprimer: 5'-TGCTAGTTATTGCTCAGCGG-3'; SEQ ID NO: 4.
[0047] The PCR reaction system is:
[0048]
[0049] The PCR reaction program was 94°C for 5min; 94°C for 30s, 52°C for 30s, 72°C for 1.5min, a total of 30 cycles; and finally 72°C for 5min.
[0050] The positive clone pET32a-HSP70 was expanded and cultivated, and the plasmid...
Embodiment 3
[0052] Induced expression of embodiment 3 fusion protein
[0053] The expression of the recombinant expression strain pET32a-HSP70 / BL21(DE3) was induced by using IPTG with a final concentration of 1mM at 37°C and 200rpm, and an empty control group of pET32a / BL21(DE3) was set at the same time; the cells were collected after 6h and boiled. SDS-PAGE analysis was carried out after the sample, after Coomassie brilliant blue staining and decolorization, the recombinant expression strain pET32a-HSP70 / BL21(DE3) was induced to obtain a fusion protein of about 70kD, and the size was in line with expectations; no protein band was obtained in the empty load, and the results were as follows figure 2 shown.
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