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Construction method and application technology of rolGLP-HV engineering bacterium

A technology of engineering strains and Escherichia coli is applied in the field of biopharmaceuticals to delay and improve thrombosis symptoms

Inactive Publication Date: 2014-06-25
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to the literature search, there is currently no combination of rolGLP-1 and HV 1 Fusion expression of two genes and report of rolGLP-HV Escherichia coli engineering strain

Method used

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  • Construction method and application technology of rolGLP-HV engineering bacterium
  • Construction method and application technology of rolGLP-HV engineering bacterium
  • Construction method and application technology of rolGLP-HV engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of a fusion gene rolGLP-HV. Including the following steps:

[0037] (1) Using pMD-GLP as a template (the gene sequence of rolGLP-1 has been applied for a national invention patent by the inventor, and the patent publication number is: CN 101824388A), obtain the rolGLP-1 gene fragment by PCR (using Pfu enzyme). The PCR primers used in the experiment were designed according to the sequence of rolGLP-1:

[0038] P1: 5′-CATTCTGAGGGTACTTTCACTTCTG-3′

[0039] P2: 5′-TCTACCATCAACCAACCAAGCGATG-3′

[0040] (2) Using gene semi-synthesis method to obtain rHV 1 Gene fragment( image 3 A). First according to rHV 1 Synthesize four oligonucleotide fragments from the gene sequence: P3, P4, P5, P6:

[0041] P3: 5′-GTTGTCTACACTGACTGTACTGAATCTGGTCAAAACTTGTGTTTGTGTGAAGGTTCCAACGTTTGTGGTCAAGGTAACAAGTGTATCTT-3′

[0042] P4: 5′-TTACTGGTGAGGGTACTCCTAAGCCACAAATCCCATAACGACGGTGACTTCGAGGAGATTCCAGAGGAATACTTGCAATAAAAGCTTGGG-3′

[0043] P5: 5′-GGCTTAGGAGTACCCTCACCAGTAACACATTGGTTT...

Embodiment 2

[0053] Construction of an Escherichia coli expression vector pNK-EC-rolGLP-HV containing rolGLP-HV fusion gene.

[0054] The rolGLP-HV gene fragment was digested with HindIII. Plasmid pET-22b(+) was digested with Nco I, blunted with Pfu enzyme, and then the large fragment of the vector was digested with HindIII. The digested vector and gene fragments were ligated ( figure 2 ). Picking positive transformants is the expression vector used in the present invention, the Escherichia coli expression vector pNK-EC-rolGLP-HV ( Figure 5 ).

Embodiment 3

[0056] Induced expression of Escherichia coli engineering strain NK-rolGLP-HV-E007 and purification of rolGLP-HV.

[0057] Select the NK-rolGLP-HV-E007 strain and culture it in a shaker flask at 37°C and 230r to OD 600 When the final concentration was 0.6-0.8, add the inducer IPTG with a final concentration of 1mmol / L, induce at 25°C for 12h, centrifuge the bacterial cells for ultrasonic disruption, and extract the protein for Tricine-SDS electrophoresis detection to prove the successful expression of rolGLP-HV( Figure 6 A). The supernatant after ultrasonic treatment was purified by affinity chromatography (Ni-NTA chromatography column) to obtain high-purity rolGLP-HV ( Figure 6 B). Western blot results ( Figure 6 C) shows that the constructed Escherichia coli has successfully expressed rolGLP-HV.

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Abstract

The invention relates to a construction and preparation method and an application technology for producing a dual-functional medicine (rolGLP-HV) engineering bacterium for treating diabetes and thromboembolic complications. The construction method disclosed herein is characterized by fusing long-acting insulinotropin rolGLP-1 having the effect of treating the symptoms and causes of type 2 diabetes with hirudin rHV1 which can effectively control cardio-cerebrovascular complications to achieve the dual functions of preventing and treating diabetes and thromboembolic complications. Specifically, by applying gene engineering technique, the fused gene rolGLP-HV of rolGLP-1 and rHV1 is obtained; by cloning the fused gene rolGLP-HV to an escherichia coli expression vector pET22b(+), an expression vector pNK-EC-rolGLP-HV is constructed; by conversing escherichia coli, the engineering bacteria NK-rolGLP-HV-E007 with highly expressing rolGLP-HV is obtained; and by conducting inducible expression and purification of the strain, the high-purity rolGLP-HV can be obtained. According to the invention, by perfusing the rolGLP-HV into the stomach of the Hu thrombus model mice, the thrombosis can be significantly slowered and alleviated; by perfusing the rolGLP-HV into the stomach of the diabete model mice, the blood glucose can be obviously reduced, and diabete symptoms can be improved, thus the rolGLP-HV disclosed herein can be used for developing the dual-function medicines for simultaneously treating diabetes and thromboembolic complications thereof.

Description

technical field [0001] The invention relates to a construction method and application technology of a rolGLP-HV (long-acting insulin-stimulating-hirudin) engineering bacterium, belonging to the technical field of biopharmaceuticals. Background technique [0002] Diabetes mellitus (diabetes mellitus) is a metabolic disorder caused by the absolute or relative insufficient secretion of insulin and the decrease of the body's sensitivity to insulin. One of the most common chronic diseases. At present, with the improvement of people's living standards, the aging population and the increase in the incidence of obesity, the incidence of diabetes is increasing year by year, and tends to be younger. According to statistics, there are 40 million diagnosed diabetic patients in China, and the number is increasing at a rate of 1 million per year. It is expected to reach 60 million by 2025. [0003] Nowadays, diabetes is said by many scholars as the "root of all diseases", because if dia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/63C12N15/70C12N1/21C07K19/00A61K38/58A61P3/10A61P7/02C12R1/19A61K38/26
Inventor 李明刚卫一鸣王维婧马百成胡晓宇张耀方乔坤艳李宠马志华
Owner NANKAI UNIV
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