Method for detecting leptospiral infection by recombined multi-epitope peptide protein
A technology for detection of leptospira and infection is applied in the field of medical immunodetection, which can solve the problems of unfavorable early rapid detection and complex antigen structure, and achieves the effect of being beneficial to popularization and application.
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Embodiment 1
[0030] Example 1. Screening of T cell and B cell dominant antigenic determinants
[0031] HLA-DR binding peptide prediction tool ProPred (http: / / www.imtech.res.in / raghava / propred / ) and EMBOSS software package (http: / / bioinfo.hku.hk / EMBOSS / ) were used to predict the hook Major T- and B-cell epitopes of the telospiral outer membrane proteins OmpL1, LipL21, and LipL32. The predicted epitope fragment was amplified by PCR method and then inserted into phage M13KE to display on the surface of PIII protein. The phage protein was purified and each epitope was identified for screening.
Embodiment 2
[0032] Example 2. Containing the construction of multi-epitope peptide vector
[0033] According to the codon preference of the expression system, the epitope segment tandem gene 1mp was artificially synthesized, in which the epitopes were connected by flexible peptide segments, and the two ends of the synthetic gene contained BamHI, BglII and EcoRI sites respectively. The synthetic gene was inserted into the pUC57 vector to construct the p-lmp vector. After the sequencing was correct, the 1mp gene was isolated by digestion with BamHI and EcoRI, and inserted into the p-1mp vector digested with BglII and EcoRI to construct the p-21mp vector. Bits are repeated twice in the vector. The 21mp fragment was separated by restriction digestion with BamHI and EcoRI and inserted into the expression vector pET28a to construct the expression vector pET28a-21mp. The pET28a-21mp recombinant plasmid can produce two bands of about 5300bp and 600bp after digestion with BamHI and EcoRI, which i...
Embodiment 3
[0036] Example 3. Induced expression of engineering bacteria pET28a-21mp / BL21
[0037] Transfer pET28a-21mp into Escherichia coli expression strain BL21(DE3) to express the target protein. Specifically, pick a single clone of pET28a-21mp and inoculate it into 3ml of LB medium, culture overnight at 37°C with shaking at 220rpm; re-inoculate the cultured bacterial solution into 3ml of LB medium at a ratio of 1:100, at 37°C, Shake culture at 220rpm for 3 hours, add IPTG with a final concentration of 1mM, induce for 4 hours at 37°C and 220rpm, collect the bacterial pellet by centrifugation; resuspend the pellet in 1×PBS buffer, perform ultrasonic disruption, centrifuge at 4°C, and take The supernatant and precipitate were analyzed by SDS-PAGE, and a specific expression band appeared at about 22kD, which was consistent with the expected molecular weight, and the target protein mainly existed in soluble form. ( figure 2 ).
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