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A kind of 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 Problems such as inconvenience of administration and risk of adverse reactions, fluctuation of blood drug concentration, etc.

Active Publication Date: 2016-06-22
杭州先为达生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In spite of this, the shortcomings such as fast plasma clearance and low bioavailability cannot be overcome, and the result of these shortcomings is that frequent injections of interferon are required to reach the effective plasma therapeutic concentration; Large fluctuations, forming peaks and valleys in drug concentration
This may increase the cost of treatment as well as the inconvenience of administration and the risk of adverse reactions

Method used

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  • A kind of lambda interferon mutant and polyethylene glycol derivative
  • A kind of lambda interferon mutant and polyethylene glycol derivative
  • A kind of lambda interferon mutant and polyethylene glycol derivative

Examples

Experimental program
Comparison scheme
Effect test

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'-GGCCCGCGTCCGCGCGGCCGT-3'

[0045] P4: 5'-CCTCGAGTTATTTATTAGGTCGATTCCGG-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 used...

Embodiment 2

[0052] Example 2: Expression and purification of IFN-λ1L62S mutant

[0053] The engineered bacteria were inoculated in LB medium containing ampicillin at a ratio of 1:100, cultured with shaking at 37°C until the OD600 of the bacterial solution was 0.4-0.6, and induced by adding a final concentration of 0.5mMIPTG, and the bacterial cells were collected after continuing to cultivate for 4h ( The SDS-PAGE pattern of the bacteria is shown in 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 ...

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, react in 25mM Tris-HCl buffer at 4°C for 10h, and adjust the pH of the reaction system with acetic acid solution to below 5.0 to terminate the reaction. The coupling degree of the reaction was detected by SDS-PAGE.

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Abstract

The invention relates to a Lambda interferon mutant. The 62nd leucine of an interferon lambda1 mutates into serine, and then the novel mutant interferon Lambda is obtained. Compared with the interferon lambda1, the mutant interferon Lambda has higher stability. The invention further relates to a polyethylene glycol derivative of the Lambda interferon mutant and the purpose of the polyethylene glycol derivative.

Description

technical field [0001] The present invention relates to IFN-λ1 mutants, polyethylene glycol derivatives thereof and preparation methods thereof, and polyethylene glycol derivatives comprising the IFN-λ1 mutants or the mutants have the functions of preventing or treating viral diseases, Drug applications such as tumors. Background technique [0002] Interferon is an important family of cytokines, with broad-spectrum anti-virus, anti-cell proliferation and immune regulation. [0003] To date, six forms of interferon have been identified, which fall into three broad groups. So-called "type I" interferons include interferon alpha, interferon beta, interferon omega, interferon delta, interferon tau. Currently, interferon gamma and a subclass of interferon alpha are the only type II interferons. Type III interferons are a recently discovered family of cytokines that include interferons λ1, λ2, and λ3, also known as IL-28A, IL-28B, and IL-29 [0004] IL-28A, IL-28B and IL-29 co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/555C07K1/107C12N15/20C12N15/70C12N1/21A61K38/21A61K47/48A61P29/00A61P31/12A61P31/04A61P35/00
CPCA61K38/00A61K47/60C07K14/555
Inventor 田硕杨璐潘海陈全民李尧
Owner 杭州先为达生物科技股份有限公司
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