Lambda interferon mutant and polyethylene glycol derivative
A polyethylene glycol, mutant technology, applied in the directions of interferon, cytokine/lymphokine/interferon, biochemical equipment and methods, etc., can solve the problem of fast plasma clearance, low bioavailability, and increased treatment costs Inconvenience of administration and risk of adverse reactions, fluctuation of blood drug concentration, etc.
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Embodiment 1
[0037] Example 1: Construction of engineering bacteria for expression of IFN-λ1L62S mutant and sequence confirmation
[0038] Extract the plasmid of IFN-λ1 as a template, and mutate the 62-position leucine (CTG) into serine (TCT). The first round of PCR includes two reaction systems:
[0039] The primers of reaction system 1 are:
[0040] P1: 5'-CCATATGGGTCCGGTGCCGACCTCGAAACCG-3'
[0041] P2: 5'-ACGGCCGCGCGGACGCGGGCC-3'
[0042] Amplify the 62 position and its upstream DNA sequence.
[0043] The primers of reaction system 2 are:
[0044] P3: 5'-GGC CCG CGT CCG CGC GGC CGT-3'
[0045] P4: 5'-CTCGAGTTATTTATTAGGTCGATTCCGG-3'
[0046] Amplify the 62 position and its downstream DNA sequence.
[0047] The PCR conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 1 min, 30 cycles, and finally extension at 72°C for 5 min.
[0048] In the second round of PCR, the product of the first round of PCR was...
Embodiment 2
[0052] Example 2: Expression and purification of IFN-λ1L62S mutant
[0053] Inoculate the engineered bacteria in the LB medium containing ampicillin at a ratio of 1:100, culture with shaking at 37°C until the OD600 of the bacteria solution is 0.4-0.6, add the final concentration of 0.5mM IPTG to induce, continue to culture for 4 hours, and collect the bacteria (For the SDS-PAGE pattern of the bacteria, see figure 1 ). When Escherichia coli BL21 (DE3) is used as the host, the expressed IFN-λ1 accounts for about 30-50% of the total bacterial protein, and mainly exists in the form of inclusion bodies.
[0054] Wash the fermented cells with TE solution (m:V=1:10) for 3 times, and then perform high-pressure homogenization and crushing. The crushing condition is: 35Mpa high-pressure homogenization. After homogenization, the bacteria breaking rate was checked under a microscope. When the bacterial cell fragmentation rate is about 95% (approximately 2 to 3 times of bacterial cell d...
Embodiment 3
[0056] Example 3: PEG conjugation of IFN-λ1L62S mutant
[0057] Mix the purified protein IFN-λ1 with mPEG-MAL with a molecular weight of 20kDa at a molar ratio of 1 / 10-1 / 5, and react in 25mM Tris-HCl buffer at 4°C for 10h, then adjust the reaction system with acetic acid solution pH to below 5.0 to terminate the reaction. The coupling degree of the reaction was detected by SDS-PAGE.
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