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Recombinant leukocyte inhibitory factor and leech peptide chimeric protein mutant

A technology of inhibitory factors and chimeric proteins, which is applied in the field of molecular biology, can solve the problems of easy mismatch, lower protein purity, and lower biological activity, so as to reduce the probability of mismatch, improve protein purity, and reduce isomers The effect of content

Pending Publication Date: 2022-06-28
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long side chain of cysteine, it is easy to produce mismatches during translation and expression, resulting in isomers that reduce protein purity and also reduce biological activity

Method used

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  • Recombinant leukocyte inhibitory factor and leech peptide chimeric protein mutant
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  • Recombinant leukocyte inhibitory factor and leech peptide chimeric protein mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Acquisition of TNHH gene mutants

[0107] 1. Primer synthesis

[0108] According to the mutation sites of TNHH shown in Table 1, Primer software was used to design the primers required for mutation, and Shanghai Sangon Engineering was entrusted to design and synthesize them. See 2 for primers required for TNHH mutants.

[0109] Table 1 Mutation sites of different TNHH mutants

[0110] mutant Mutation site TNHH-M1 278 Pro→Thr, 282 Leu→Glu, 162, 214 Cys→Ala TNHH-M2 274th Phe→Ser, 277th Ile→Gly, 162nd, 214th Cys→Ala TNHH-M3 278 Pro→Thr, 282 Leu→Glu, 211, 214 Cys→Ala TNHH-M4 274th Phe→Ser, 277th Ile→Gly, 211th, 214th Cys→Ala

[0111] Table 2 Primers required for TNHH mutants

[0112]

[0113]

[0114] 2. PCR reaction

[0115] The primers required to mutate the 162nd amino acid of the original TNHH sequence, the template DNA and the buffer system were mixed according to the following reaction system.

[0116] ...

Embodiment 2

[0150] Example 2 Shake flask expression of mutant pET-3c-TNHH / BL21(DE3)pLysS-M1~pET-3c-TNHH / BL21(DE3)pLysS-M4 strains

[0151] The four mutant strains and the original E. coli strain pET-3c-TNHH / BL21(DE3)pLysS were used for preliminary expression studies on shake flasks. First, pick a single clone from the corresponding plate of the strain and inoculate it into LB ampicillin medium (0.5% yeast powder, 1% peptone, 1% sodium chloride, 100 mg / L ampicillin), inoculate 2 bottles per strain, and cultivate at 37 °C for 10 h. OD 600 Between 0.8 and 1.2, use a final concentration of 0.4 mol / LIPTG to induce expression, and the induction for 4 hours ends. The bacterial solution was centrifuged at 12,000 rpm for 3 min to discard the supernatant, and its expression was analyzed by SDS-PADE. The results are shown in figure 1 ,from figure 1 It can be seen that the protein expression level after gene mutation is increased compared with the initial TNHH expression level.

Embodiment 3

[0152] Example 3 Fermentation and purification of TNHH and mutants TNHHM1-M4

[0153] 1. Fermentation induction of original strain pET-3c-TNHH / BL21(DE3)pLysS and mutant strain pET-3c-TNHH / BL21(DE3)pLysS-M1~pET-3c-TNHH / BL21(DE3)pLysS-M4 Express

[0154] 1. Seed cultivation:

[0155] Pick the original strain pET-3c-TNHH / BL21(DE3)pLysS, and the mutant strain pET-3c-TNHH / BL21(DE3)pLysS-M1~pET-3c-TNHH / BL21(DE3)pLysS-M4 single colonies respectively It was inoculated into LB medium containing 100 mg / L ampicillin, and cultured at 37° C. and 150 rpm for 10 h to obtain seed liquid for fermentation.

[0156] 2. 30L fermentation tank fermentation:

[0157] Add 10L fermentation base medium (0.3% yeast powder, 0.5% peptone, 3.5% dodecahydrate and disodium hydrogen phosphate, 0.5% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.1% sodium chloride, 0.1% magnesium sulfate, 1% glucose), autoclaved, after the medium was cooled to 37°C, the pH was adjusted to 7.0 with ammonia water,...

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Abstract

The invention belongs to the field of molecular biology, and particularly discloses a recombinant leukocyte inhibiting factor and leech peptide chimeric protein mutant. On the basis of TNHH, cysteine which easily causes translation expression mismatch is mutated, and part of non-polar amino acid at a thrombin binding part is mutated into other amino acid, so that the TNHH mutant is obtained. The TNHH is a chimeric protein with double functions, two problems are simultaneously solved through a gene site-directed mutagenesis technology, the content of isomers is reduced, the purity of the protein is improved, the biological activity of the TNHH is greatly improved, and the TNHH has very high practical significance.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a recombinant leukocyte inhibitory factor and a leech peptide chimeric protein mutant. Background technique [0002] In my country, the mortality rate of cerebrovascular diseases is second only to malignant tumors, and the mortality rate caused by ischemic brain injury accounts for 2 / 3 of them. There are two main types of ischemic stroke: one is stroke caused by cerebral atherosclerosis or cerebrovascular embolism, and the other is stroke caused by cerebral blood flow hypoperfusion caused by cardiac arrest, etc. At present, thrombolysis is mainly used for treatment, and anti-platelet aggregation drugs such as aspirin and clopidogrel are used for treatment. Although it can improve the situation of stroke, the treatment cycle is long and there are serious sequelae, such as hemiplegia and cognitive impairment. And the treatment is expensive and has many side effects. [0003] Stu...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/19A61K38/58A61P9/00A61P9/10C12R1/19
CPCC07K14/52C07K14/815C12N15/70A61P9/00A61P9/10C07K2319/00A61K38/00
Inventor 张贵民张冰雪全艳彩刘忠
Owner LUNAN PHARMA GROUP CORPORATION
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