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Constructing method for prokaryotic expression vector for producing heparinase I at high yield

A construction method and prokaryotic expression technology, applied in the field of genetic engineering, can solve the problems of low molecular weight heparin production technology research, low yield of wild bacteria, limited sources of heparinase, etc., and achieve good application prospects

Inactive Publication Date: 2012-08-01
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of using enzymatic hydrolysis to produce low-molecular-weight heparin are: (1) On the one hand, the source of heparinase is limited, the yield of wild bacteria is very low, and expensive heparin is required for induced expression, resulting in high production costs
my country is a big exporter of heparin raw materials, but has not been able to conduct systematic research on the production technology of low molecular weight heparin

Method used

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  • Constructing method for prokaryotic expression vector for producing heparinase I at high yield
  • Constructing method for prokaryotic expression vector for producing heparinase I at high yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Acquisition of Heparanase Ⅰ Gene Sequence of Flavobacterium Heparinus

[0017] According to the gene sequence of heparanase Ⅰ, the following primers were designed:

[0018] F1: 5’-TAGAATTCCAGCAAAAAAAAATCCG-3’

[0019] R1: 5'-GGCAAGCTTGTCTGGCAGTTTCGCTGTA-3'

[0020] Use PCR reaction to amplify the gene sequence encoding heparanase Ⅰ, the PCR reaction system is (50μl): 10×Buffer 5μl, MgCl 2 3 μl, dNTP 1 μl, DNA 1 μl, Taq enzyme 0.5 μl, primer mix 2 μl, double distilled water 37.5 μl. The reaction parameters are: 97°C for 5min, (97°C for 50s, 55°C for 50s, 72°C for 1min) for a total of 30 cycles, and finally extended at 72°C for 10min, agarose gel electrophoresis to recover the PCR product, and the recovered product was sent to Shanghai Sangon Bioengineering Co., Ltd. sequencing, the obtained sequence is shown in SEQ ID NO.1.

Embodiment 2

[0021] Example 2. Construction of recombinant Escherichia coli expression vector

[0022] use Eco RI and Hin dⅢ double-enzyme digest the PCR amplification product and pET-28a plasmid DNA respectively, recover the target band by agarose gel electrophoresis, and use T 4 DNA ligase ligated the recovered target bands to obtain the prokaryotic expression vector pET28a-HpaI, and used CaCl 2 Transform it into Escherichia coli BL21 by heat shock at 42°C for 90 seconds. Single colonies were screened for Kana resistance. The selected transformed clones were verified by PCR, enzyme digestion and DNA sequencing to confirm that the clones were correct and then sent to Shanghai Bioengineering Co., Ltd. for sequencing. See the specific construction process figure 1 .

Embodiment 3

[0023] Example 3. Transformation and screening of recombinant Escherichia coli expression vector

[0024] The correctly identified recombinant expression vector pET28a-HpaI plasmid was transformed into Escherichia coli BL21 strain, and 0.5mmol / L IPTG was added to induce expression for 9h, the cells were resuspended in phosphate buffer, and the heparin crude enzyme solution was obtained by ultrasonication. Heparanase activity was measured by Azure A method, and the strain HpaⅠ with the highest expression of heparinase activity was screened out (47).

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Abstract

The invention discloses a constructing method of prokaryotic expression vector for producing heparinase I at high yield. The constructing method comprises the following steps of: taking F1:5'-TAGAATTCCAGCAAAAAAAATCCG-3' and R1:5'-GGCAAGCTTGTCTGGCAGTTTCGCTGTA-3' as primers; carrying out PCR (Polymerase Chain Reaction) to obtain a gene sequence of an encoded heparinase I, which is shown as in an SEQIDNO.1; connecting the gene sequence of the encoded heparinase I to a pET-28a vector to obtain an a recombinant expression vector pET28a-HpaI; and transferring the expression vector pET28a-HpaI plasmid into an Escherichia coli BL21 strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a method for constructing a prokaryotic expression vector with high yield of heparanase I. Background technique [0002] Heparanase (heparanase, Hpa) is a type of D-glycosidase that has been studied more in recent years. It not only exists in normal tissues, such as placenta and lymphoid organs, but also affects the formation of embryos, the formation of new blood vessels, The development of the nervous system, inflammatory response, etc. all play normal physiological functions; at the same time, they also exist on the surface of various malignant tumor cells, which can promote cell invasion and metastasis, induce tumor angiogenesis, and thus reduce the survival rate of tumor patients. At present, the focus of research is how to inhibit the heparanase activity expressed on the surface of tumor cells, so as to effectively treat tumors. Heparinase has a wide range of uses, and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/60C12N1/21
Inventor 于平
Owner ZHEJIANG GONGSHANG UNIVERSITY
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