Preparation method and application of treponema pallidum TP47
A TP47-R, TP47-F technology, applied in the fields of biomedicine, infectious disease diagnosis and detection, can solve problems such as poor stability
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Embodiment 1
[0086] Example 1 Construction and expression purification of recombinant wild-type TP47 antigen (WTP47)
[0087]The 19th-434th amino acid of TP47 was selected as the antigen region, and the codon-optimized gene sequence 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] Transform pET28a-WTP47 into Escherichia coli E. coli BL21 (Rosetta) competent cells, and induce expression according to the method described in the Molecular Cloning Experiment Guide. Specifically, culture at 37°C at 200 r / min until OD600=0.8, add IPTG to a final concentration of 1 mM, and induce at 37°C for 4 hours. The clone that induced the target protein was named R-pET28a-WTP47. R-pET28a-WTP47 was mass-fermented according to the induction conditions determined...
Embodiment 2
[0092] Example 2 Construction and expression purification of recombinant truncated DTP47 antigen
[0093] Select amino acids 26-410 of TP47 as the antigen region, use the nucleotide sequence SEQ ID NO: 5 of the wild-type WTP47 in Example 1 as a template, and use 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 performing 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] Transform pET28a-DTP47 into Escherichia coli E.coli BL21 (Rosetta) competent cells, and induce expression according to the method described in the Molecular Cloning Experiment Guide. Specifically, culture at 37°C at 200 r / min until OD600=0.8, add IPTG to a final concentration of 1 mM, and induce at 37°C for 4 hours. The clone that induced the target pr...
Embodiment 3
[0098] Example 3 Construction and Expression Purification of Recombinant Truncated and Mutant C296S Antigen
[0099] Select the 26th-410th amino acid of TP47 as the antigen region, use the nucleotide sequence SEQ ID NO: 6 of DTP47 in Example 2 as a template, and use 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-lapPCR amplification and gel recovery to obtain nucleosides according to the method described in the Molecular Cloning Experiment Guide 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. Transform pET28a-C296S into Escherichia coli E.coli BL21 (Rosetta) competent cells, and induce expression according to the method described in the molecular cloning experiment guide. Specifically, culture at 37°C at 200 r / min until OD600=0.8, add IPTG to a final concentration of 1 mM, and i...
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