A kind of recombinant plasmid pet28a-ag43, surface display plasmid, recombinant engineering bacteria and application thereof
A technology of recombinant engineering bacteria and surface display, which is applied in the field of genetic engineering, can solve the problems of limiting the application of cell surface display technology, and achieve the effects of rapid response, improved enzyme protein expression activity, and thorough reaction
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[0039] The present invention provides a method for preparing the recombinant plasmid described in the above scheme, comprising the steps of:
[0040]1) Using EcoRI and HindIII to perform double enzyme digestion on the genes encoding plasmid pET28a and Ag43, respectively, to obtain pET28a after digestion and the gene encoding Ag43 after digestion;
[0041] 2) Ligate the digested pET28a and the digested Ag43 coding gene in step 1) to obtain the recombinant plasmid pET28a-Ag43.
[0042] In the present invention, EcoRI and HindIII are used to carry out double enzyme digestion on the coding genes of plasmid pET28a and Ag43 respectively, so as to obtain pET28a after digestion and Ag43 after digestion. In the present invention, the preparation method of the Ag43-encoding gene preferably includes the following steps:
[0043] a. amplifying and culturing Escherichia coli K-12 MG1655 to obtain Escherichia coli K-12 MG1655 cells;
[0044] b. extracting the total DNA of the Escherichia ...
Embodiment 1
[0068] Escherichia coli K-12 MG1655 was amplified and cultured in LB medium at 37°C for 16 hours to obtain Escherichia coli K-12MG1655 cells. The obtained Escherichia coli K-12 MG1655 cells were centrifuged at a speed of 4000 rpm for 10 min, and the precipitate was collected, which was Escherichia coli. The total DNA of Escherichia coli K-12MG1655 was extracted by liquid nitrogen grinding method at a speed of 300 rpm for 5-10 min. Using the total DNA of Escherichia coli K-12 MG1655 as a template, the Ag43 coding gene was amplified using primers Ag43-EcoRI-F and Ag43-HindIII-R (amplification conditions: 94°C, 4min; 94°C, 1min; 56°C , 30s; 72°C, 1kb / min; 29 cycles; 72°C, 10min. The amplification system is: genomic DNA or plasmid DNA 10-100ng, front-end primer (10μM) 2-3μL, back-end primer (10μM)
[0069] 2~3μL, dNTP mixture (10μM) 1~3μL, 10×Ex Taq buffer (Mg 2+ ) 5 μL, Ex Taq polymerase 0.5 μL, the total system is 50 μL. The gene encoding Ag43 was obtained.
[0070] EcoRI an...
Embodiment 2
[0073] Use continuous overlap extension PCR to excise 53-137aa or 53-699aa of the Ag43 gene in the recombinant plasmid pET28a-Ag43 obtained in Example 1, target gene fragment 100ng, 10×QuickCut buffer 5 μL, QuickCut enzyme I 1-2 μL, QuickCut Enzyme II 1~2μL, the total system is 50μL. Insert the multiple cloning site MC for ligation; the reaction system is 3:1 mass ratio of enzyme-digested target gene fragment to enzyme-digested plasmid DNA, 10×T4 DNA ligation buffer 2-3 μL, T4 DNA ligase 2-3 μL, total system 20 μL. The surface display plasmids pET28a-Ag43-138 and pET28a-Ag43-700 were obtained.
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