Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof
A technology of human serum albumin and serum albumin, which is applied in the direction of peptide/protein components, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of low efficiency and high production cost, extend the half-life and reduce the burden of treatment , Improve the effect of bioavailability and therapeutic value
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Embodiment 1
[0053] Embodiment 1: human albumin gene cloning
[0054] The human HSA mature peptide gene was prepared by RT-PCR, and the primers were designed as follows:
[0055] HSA1:5'-GATGCACACAAGAGTGAGGTGCTCA-3',
[0056] HSA2: 5'-AAGCTAGCTT ATAAGCCTAA GGCAGCTTG ACTTGC-3'.
[0057] Human fetal liver tissue was taken, and total RNA was prepared by the Trizol method. RT-PCR amplification: In a 0.2mL PCR tube, add 25μL of 2×RT PCR buffer, 3μL of total RNA (50ng), primers HSA1 0.5μL (25μM), HSA2 0.5μL (25μM), RT-Phusion mixed enzyme 1 μL, add DEPC water to a final volume of 50 μL. PCR conditions: 45°C for 30min; then 94°C for 2min; then 94°C for 30sec; 55°C for 1min, 72°C for 2min, a total of 30 cycles. Finally, keep warm at 72°C for 5 minutes. The PCR product was subjected to agarose gel electrophoresis to observe the amplification result and product size. Then use the gel recovery method to purify the HSA gene fragment. The specific method is to cut off the DNA band with a molecula...
Embodiment 2
[0058] Embodiment 2: Exendin-4 gene synthesis
[0059] The Exendin-4 gene is prepared by artificial synthesis. The sequence according to SEQ ID NO: 5 was synthesized from the Exendin-4 gene of Shanghai Sangon Bioengineering Technology Service Co., Ltd., and a StuI restriction site was provided at the 5' end.
Embodiment 3
[0060] Embodiment 3: Construction and cloning of Exendin-4-HSA fusion gene
[0061] In this experiment, the Exendin-4 and HSA genes were directly fused to form the Exendin-4-HSA fusion gene by overlapping PCR method as an example to illustrate the method of synthesizing the fusion gene. The specific method is as follows: without adding primers and polymerase, mix the components according to the following system.
[0062] 5×phusion HF buffer 20 μL, dNTPs (10 mM) 2 μL, Exendin-4 DNA fragment 2 μL, HSA DNA fragment 2 μL, ddH 2 O 69 μL for a total volume of 95 μL.
[0063] Denature at 99°C for 10 min, then cool naturally at room temperature. Add 0.5 μL of Phusion high-fidelity enzyme, incubate at 72°C for 15 minutes, and then add primers
[0064] Ex1: (5′-TA AGGCCT CACGGTGAAGGTACTTTCACTT-3′,Stu1) (50 μM) and
[0065] HSA2: (5′-AAGCTAGCTTATAAGCCTAAGGCAGCTTGACTTGC-3′, Nhe1) (50 μM) 2 μL each, Phusion high-fidelity enzyme 0.5 μL, for conventional PCR amplification:
[0066] Pr...
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