Preparation and Application of Recombinant Antigen of Treponema pallidum tp47
A TP47-R, TP47-F technology, applied in the field of biomedicine, infectious disease diagnosis and detection, can solve the problems of poor stability, achieve excellent thermal stability, good soluble expression, and improve the effect of solubility
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Embodiment 1
[0086] Example 1 Construction, expression and purification of recombinant wild-type TP47 antigen (WTP47)
[0087]The amino acids at positions 19-434 of TP47 were selected as the antigenic region, and the gene sequence after codon optimization was chemically synthesized to obtain the nucleotide sequence WTP47 (SEQ ID NO: 5). The WTP47 gene fragment was constructed into the pET28a vector according to the method described in the Molecular Cloning Experiment Guide (fourth edition), and the recombinant vector was named pET28a-WTP47.
[0088] The pET28a-WTP47 was transformed into E. coli BL21 (Rosetta) competent cells, and expression was induced according to the method described in the molecular cloning experiment guide. Specifically, the cells were cultured at 37°C at 200 r / min until OD600 = 0.8, IPTG was added to a final concentration of 1 mM, and the cells were induced at 37°C for 4 hours. The clone that induced the target protein was named R-pET28a-WTP47. R-pET28a-WTP47 was su...
Embodiment 2
[0092] Example 2 Construction, expression and purification of recombinant truncated DTP47 antigen
[0093] The 26-410th amino acids of TP47 were selected as the antigenic region, the nucleotide sequence SEQ ID NO: 5 of the wild-type WTP47 in Example 1 was used as the template, and the primer pair TP47-F (SEQ ID NO: 9), TP47- R (SEQ ID NO: 10), the nucleotide sequence DTP47 (SEQ ID NO: 6) was obtained by PCR amplification and gel recovery according to the method described in the molecular cloning experiment guide. The DTP47 gene fragment was constructed into the pET28a vector, and the recombinant vector was named pET28a-DTP47.
[0094] The pET28a-DTP47 was transformed into E. coli BL21 (Rosetta) competent cells, and expression was induced according to the method described in the molecular cloning experiment guide. Specifically, the cells were cultured at 37°C at 200 r / min until OD600 = 0.8, IPTG was added to a final concentration of 1 mM, and the cells were induced at 37°C for...
Embodiment 3
[0098] Example 3 Construction, expression and purification of recombinant truncated and mutant C296S antigen
[0099] The amino acids at positions 26-410 of TP47 were selected as the antigenic region, the nucleotide sequence SEQ ID NO: 6 of DTP47 in Example 2 was used as the template, and the primer pair TP47-F (SEQ ID NO: 9), C296S-R (SEQ ID NO: 12); C296S-F (SEQ ID NO: 11), TP47-R (SEQ ID NO: 10) were subjected to over-lap PCR amplification and gel recovery according to the method described in the molecular cloning experiment guide to obtain nucleosides Acid sequence C296S (SEQ ID NO: 7).
[0100] The C296S gene fragment was constructed into the pET28a vector, and the recombinant vector was named pET28a-C296S. The pET28a-C296S was transformed into E. coli BL21 (Rosetta) competent cells, and expression was induced according to the method described in the molecular cloning experiment guide. Specifically, the cells were cultured at 37°C at 200 r / min until OD600 = 0.8, IPTG wa...
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