A kind of aav carrier for treating type 2 diabetes and its preparation method and application
A type 2 diabetes and carrier technology, applied in the biological field, can solve the problems of high economic cost, low patient compliance, poor application convenience, etc., and achieve good safety, low immunogenicity, and increased shelf life
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Embodiment 1
[0031] Embodiment 1 prepares the experimental sequence containing GLP-1 gene
[0032] First, the nucleotide sequence containing the GLP-1 gene was obtained through chemical synthesis by Yingwei Jieji Company. The nucleotide sequence included: a 141bp signal peptide sequence and a 96bp GLP-1 gene sequence. A 6bp restriction site sequence (5'-TCTAGA; and GGATCC-3') and a 2bp protective base were added to the 5' and 3' ends respectively, resulting in a total of 253bp nucleotide sequence, the nucleotide The sequence is shown as SEQ ID NO: 1 in the sequence listing.
[0033] Utilize the PCR method to amplify and enrich the above-mentioned nucleotide sequence, and the specific primers used to amplify the nucleotide sequence are respectively:
[0034] GLP-1-5': 5'-CGTCTAGAATGAAGATTATCCTGTGGC-3' (as shown in SEQ ID NO: 2 in the sequence listing);
[0035] GLP-1-3': 5'-ATGGATCCTTATCCTCGGCCTT-3' (shown as SEQ ID NO: 3 in the sequence listing).
[0036] The PCR reaction system is: in ...
Embodiment 2
[0038] The subcloning of embodiment 2GLP-1 gene
[0039] The nucleotide fragment of the purified GLP-1 gene prepared in Example 1 was ligated with the pEasy blunt vector (purchased from TransGen Biotech) to construct the pEasy blunt-GLP-1 vector. Firstly, the pEasyblunt vector was digested once with BamHI and XbaI, and then the nucleotide fragment of the purified GLP-1 gene obtained in Example 1 was also digested with BamHI and XbaI. Please refer to the conditions for the double digestion. Refer to the instruction manual of the restriction enzyme.
[0040] The pEasy blunt vector and the nucleotide fragment of the GLP-1 gene obtained after the double enzyme digestion were subjected to a ligation reaction. The connection system and method are as follows: take 1 μl of the pEasy blunt vector after double digestion, take 3 μl of the nucleotide fragment of the GLP-1 gene after double digestion, H 2 O 1 μl, solution I 5 μl, react at 16°C for 30 minutes. The obtained ligation produ...
Embodiment 3
[0041] The construction of embodiment 3pAAV-GLP-1 vector
[0042] Use restriction endonuclease BamHI, Xba I double digestion embodiment 2 to prepare gained pEasy blunt-GLP-1 vector and pAAV-MCS vector (this vector is purchased from life technologies company), the plasmid map of pAAV-MCS vector is as follows figure 1 as shown, figure 1 is the plasmid map of pAAV-MCS. Digestion to obtain the restriction fragment GLP-1(B / x) of the GLP-1 gene and the restriction fragment pAAV-(B / x) of the pAAV-MCS vector, and the obtained restriction product GLP-1(B / x) with Purification of pAAV-(B / x) (the purification kit used was purchased from Axygen, the product number is AP-GX-50), the recovered and purified product was ligated with T4 DNA ligase, ligated at 16°C for 4h to overnight, and pAAV- GLP-1 vector. Wherein the BamHI, XbaI double enzyme digestion system is as follows:
[0043]
[0044] Reaction conditions: incubate at 37°C for 4h.
[0045] Wherein the connection system is as fo...
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