A recombinant vector for expressing soluble pig intestinal trefoil peptide, construction method, recombinant engineering bacteria and expression method thereof
A technique of pig intestine trefoil peptide and recombinant engineering bacteria
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Embodiment 1
[0088] Cloning of embodiment 1 porcine intestinal trefoil peptide TFF3 gene
[0089] 1. Design and synthesis of primers for porcine intestinal trefoil peptide TFF3 gene cloning
[0090] Log in to NCBI, search out the porcine intestinal trefoil peptide TFF3 gene sequence in the porcine EST database, use DNAStar to design cloning primers including the complete ORF (see Table 1-1), and add HindIII and XhoI to the upstream and downstream of the target fragment respectively restriction endonuclease sites. The designed primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0091] Table 1-1 Primers used for TFF3 cloning
[0092]
[0093] 2. Extraction and detection of total RNA from porcine intestinal mucosa
[0094] The present invention adopts TRIZOL method to extract intestinal total RNA, and the method is as follows:
[0095] (1) Take out the preserved intestinal mucosal tissue from liquid nitrogen, quickly place it on tin foil paper, take about 100 mg, put...
Embodiment 2
[0111] The transformation of embodiment 2 plasmid pET28a
[0112] 1. Design and synthesis of human Galectin-1 gene and three pairs of primers required for cloning
[0113] Log in to NCBI, search for the human Galectin-1 gene sequence in the human EST database, use DNAStar to design the cloning primers Primera and Primerb including the complete ORF (see Table 1-2), and add HindIII, Primerb to the upstream and downstream of the target fragment, respectively. BamHI restriction endonuclease site. At the same time, the other three pairs of primers required were designed, which have a common upstream primer PrimerF1 (see Table 1-2), and NcoI and HindIII restriction endonuclease sites were added to the upstream and downstream of the obtained product, respectively. The designed primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0114] Table 1-2 Primers used for cloning
[0115]
[0116]
[0117] 2. Cloning and purification of human Galectin-1 gene
[0118...
Embodiment 3
[0179] Example 3 Construction of recombinant expression plasmid pET28a-TFF3
[0180] 1. Both the intestinal trefoil peptide TFF3 gene in Example 1 and the transformed plasmid pET28a in Example 2 were digested with HindIII and XhoI.
[0181] pET28a modified plasmid digestion system:
[0182] Configure the above enzyme digestion system, mix well, and digest at 37°C for more than 2 hours. The digested products were subjected to 1.0% agarose gel electrophoresis, and the digested products were purified and recovered with Sangon SanPrep Column DNA Gel Recovery Kit. The absorbance value and concentration were detected with a UV spectrophotometer.
[0183] TFF3 enzyme digestion system:
[0184] Configure the above enzyme digestion system, mix well, and digest at 37°C for more than 2 hours. The digested products were subjected to 3.0% agarose gel electrophoresis, and the digested products were purified and recovered with Sangon SanPrep Column DNA Gel Recovery Kit. The absorban...
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