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Recombinant thymus pentapeptide structural analogs, its production and use

A technology similar in structure to thymosin, applied in the field of genetic engineering, can solve problems such as the inability to realize the efficient preparation of cyclic peptides, and achieve the effect of low cost and high efficiency

Inactive Publication Date: 2007-06-20
广州达信生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional method of first expressing and purifying the fusion protein containing the target polypeptide, and then cleaving the target polypeptide from the fusion protein cannot achieve efficient preparation of cyclic peptides

Method used

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  • Recombinant thymus pentapeptide structural analogs, its production and use
  • Recombinant thymus pentapeptide structural analogs, its production and use
  • Recombinant thymus pentapeptide structural analogs, its production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Expression of recombinant thymopentin structural analog

[0033] 1. Synthesis of recombinant thymosin gene and construction of pTW-TP plasmid

[0034] Two complementary primers were synthesized: primer 1: AAC TGC CGT AAA GAT GTG TAT; primer 2: GCA ATA CAC ATC TTT ACG GCA; the recombinant thymosin gene with sticky ends was artificially synthesized by primer denaturation and renaturation; The cut pTWIN1 vector was ligated to obtain the recombinant expression plasmid pTW-TP. Its sequence diagram is shown in 2.

[0035] 2. Expression and purification of recombinant fusion protein

[0036]The expression plasmid was transformed into the expression host strain E.coli Strain ER2566, and the expression engineered strain pTW-TP-ER2566 was constructed, as shown in Figure 3. Pick the monoclonal shaking bacteria and culture overnight, inoculate 1L LB medium containing 100mg / L ammonium biline at 1:20, shake the bacteria at 37°C until the OD is 0.5-0.8, add IPTG to the...

Embodiment 2

[0039] Example 2 The carbon particle clearance test detects the activity of cyclo in promoting the phagocytosis of macrophages

[0040] cyc-TP and chemically synthesized linear pentapeptide TP-5 (Hainan Zhonghe Pharmaceutical) were intraperitoneally injected into NIH mice (20 g) at a dose of 5 μg / kg, and injected with a blank vehicle as a control, administered once a day for 10 consecutive days . Phagocytosis rate and phagocytosis index were determined and calculated according to literature methods. The results were tested by t.

[0041] The results (as shown in Table 1) showed that the phagocytosis rate (K) and phagocytosis index (a) of cyc-TP and TP-5 were significantly higher than that of the blank solvent control group, and had the effect of significantly improving the clearance ability. Meanwhile, the phagocytic ability of cyc-TP group was higher than that of TP-5 group.

[0042] Table 1: Effect of cyc-TP on washout capacity

[0043] Group

[0044] a: P<0.01...

Embodiment 3

[0045] Example 3 Mouse hemolytic plaque detection activity of cyclo promoting B cell function

[0046] The grouping of the mice, the dosage and the way of administration were the same as before, and the administration was performed once a day for 10 consecutive days, and a blank vehicle was injected as a control. On the 7th day of administration, each mouse was intraperitoneally injected with 0.2ml of 20% SRBC physiological saline mixed solution for immunization. On the second day after the last administration, hemolyzed plaque PFC was measured according to the literature method, and the results (see Table 2 ) through the t-test. The results showed that both cyc-TP and TP-5 effectively promoted the activity and function of B cells, and the activity of cyc-TP was stronger than that of TP-5.

[0047] Table 2: Effect of cyc-TP) on hemolytic plaque formation

[0048] Group

[0049] a: P<0.01, Experimental group vs Control; b: P<0.01, cyc-TP group vs TP-5 group.

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Abstract

Recombinant thymus pentapeptide structural analogs, its production, expression and use for preparing related immune diseases medicines are disclosed. The structural formula is cyclo-(Cys-Arg-Lys-Asp-Val-Tyr-), and it consists of Cys-guided circular thymic peptide and 6 amino acids. It has better biological activity and stability.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant thymosin analogue and a preparation method thereof, and the application of the recombinant thymosin analogue in medicine for treating immune-related diseases. Background technique [0002] U.S. Patents (U.S.Pat.Nos.4,190,646 and U.S.PaT.Nos.4,261,886) disclose artificially synthesized pentapeptides that have the same activity as human natural thymosin II (composed of 74 amino acids), and the sequence is H-ARG-LYS -ASP-VAL-TYR-OH, known as thymopentin (thymopentin or thymopoietin, TP); US patents (U.S. Pat. Nos. 4,002,740 and 4,077,949) reveal that thymopentin can selectively activate T cell differentiation, 1978 M.E. Weksler and others published an article in "Japanese Applied Medicine", claiming that thymopentin has a significant immune regulation function and has great medical application value. [0003] Since then, a number of patents in the United S...

Claims

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Application Information

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IPC IPC(8): C07K7/64C12N15/12C12P21/02C12N15/63A61K38/12A61P37/00A61P37/02A61P31/10A61P31/12
CPCY02A50/30
Inventor 韩为跃张仁怀杨立明阳勇刘德林林佶侃闻亚磊何凯
Owner 广州达信生物技术有限公司
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