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Survivin mutant containing HIV transduction structural area and its preparation method and uses

A mutant and survivin technology, which is applied in the field of survivin mutants and its preparation, can solve the problems of inefficient entry of protein macromolecules, reproductive toxicity, gene mutation, etc., and achieve the effect of avoiding reproductive toxicity and gene mutation

Inactive Publication Date: 2008-10-08
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, directly using engineered viruses to carry therapeutic genes into the body faces many problems in terms of effectiveness, operability and safety, especially the problems of gene mutation and reproductive toxicity, which restrict the clinical application of Survivin (Thr34Ala) gene therapy. Applications
[0005] Using Survivin (Thr34Ala) protein as a therapeutic drug is a direction to overcome the defects of gene therapy. However, protein macromolecules cannot efficiently enter cells, which is a long-standing technical problem in this field.

Method used

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  • Survivin mutant containing HIV transduction structural area and its preparation method and uses
  • Survivin mutant containing HIV transduction structural area and its preparation method and uses
  • Survivin mutant containing HIV transduction structural area and its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Construction of pREST-B-TAT(m)-Survivin(T34A) vector system

[0083] Total RNA was extracted from B-Cap-37 with Trizol reagent, and the integrity of RNA was identified by denaturing agarose gel electrophoresis. Use the extracted total RNA with primers Pb1: 5′-TAG AAT TCG GCC GGC TGC GGG GCA TTC GC-3′ and P2: 5′-GTC GAA TTC TCA CAG GTC TGA GCA GCG ATC CTG CTT GCT-3′, reverse Record it as cDNA, use it as a template, and amplify by PCR to obtain the amplified product which is the coding sequence of the human Survivin gene, and put the Survivin segment into the pEGM-T vector. Using this as a template, use the Survivin mutation primer Pm1: 5'-CTTGGAGGGCTGCGCCTGC GCC CCGGAGCGGATGGCCGAGGC-3'Pm2:5'-GCCTCGGCCATCCGCTCCGG GGC GCAGGCGCAGCCCTCCAAG-3' site-directed mutation of Survivin34 Thr to Ala by recombination method, the mutated Survivin gene was loaded into pEGM-T vector, and the sequence was correct. Use the HIV-TAT protein transduction sequence to add primers...

Embodiment 2

[0084] Example 2. Transformation of Escherichia coli with pREST-B-TAT(m)-Survivin(T34A) vector

[0085] Escherichia coli BL21 (DE3) competent preparation method according to "Molecular Cloning" (Science Press, second edition) CaCl 2 method, take 50 μl / tube of competent cells from the -80°C refrigerator, and after thawing the frozen strain at room temperature, add 1 μl of the correctly constructed pREST-B-TAT(m)-Survivin(T34A) expression vector, and gently rotate to mix Put the contents in an ice bath for 30 minutes, immediately transfer to a 42°C water bath for 2 minutes, then add 0.5ml of LB medium without antibiotics to each tube, place it in a 37°C water bath for 15 minutes, and shake gently on a 37°C shaker For 45 minutes, take 100 μl of transformed bacteria, spread evenly on the agar plate medium containing 100 mg / ml ampicillin, dry at room temperature, and incubate overnight at 37°C.

Embodiment 3

[0086] Example 3. Small expression of TAT(m)-Survivin(T34A) fusion protein

[0087] Pick 7 clones from the LB (containing AMP resistance) plate and culture them in 50ml LB culture medium (0.5% yeast powder, 1% peptone, 1% NaCl, Ph7.0, 0.5% AMP), 37oC, culture under 250rpm to O.D.600 = 0.6. Add 1mmol IPTG to induce protein expression. Cultivate at 37oC and 250rpm for 4 hours, collect the cells by centrifugation, and take a small amount for SDS-polyacrylamide gel electrophoresis. The results are as follows: Figure 2 Show.

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Abstract

The present invention belongs to the field of bioengineering, and is especially one kind of Survivin point mutant fusion protein gene TAT(m)-Survivin (T34A) and its recombinant expression vector, engineering bacterium containing the expression vector and its preparation, and the preparation and application in tumor treating medicine of the TAT(m)-Survivin (T34A) expression product. Through cloning, mutating, restriction, enzyme linking and other gene operating processes, recombinant TAT(m)-Survivin (T34A) fusion protein gene is obtained, recombinant expression vector is constituted, and recombinant expression vector is transformed to colibacillus to obtain the engineering bacterium expressing the recombinant TAT(m)-Survivin (T34A) fusion protein. The engineering bacterium is processed through serial steps to obtain the destination fusion protein. The present invention has obvious effect of promoting cancer cell apoptosis.

Description

technical field [0001] The invention belongs to the field of bioengineering and pharmacy, and in particular relates to a Survivin mutant containing HIV protein transduction domain and a preparation method thereof. Background technique [0002] Survivin (Survivin) was reported in 1997 (Ambrosini G, et al.Nat Med.1997, 3 (8): 917-921), and Aitieric DC of Yale University etc. utilized effector cell protease receptor ECR-1 (Effector cell Proteasereceptor-1, EPR-1) is a newest member of the IAP (Inhibitor of Apoptosis Protein) family that was first screened and isolated in the human genome library. Survivin is the smallest IAP cloned so far. It inhibits apoptosis by blocking regulatory pathways such as cysteine ​​proteases Caspase-3 and Caspase-7, so that some transformed cells cannot be cleared from the surveillance of the body's immune system, thereby Abnormal survival, accumulation, formation of dominant clones to participate in tumorigenesis. Unlike other apoptosis inhibito...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/47C07K7/06C12N15/62C12N15/63A61K38/17A61P35/00
Inventor 魏东芝马兴元郑文云马昱澍刘清海杨胜利
Owner EAST CHINA UNIV OF SCI & TECH
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