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Hyaluronidase expression vector and application thereof

A technology of hyaluronidase and expression vector, applied in the direction of application and introduction of foreign genetic material and enzyme by vector, can solve the problems of complex composition, immune response of patients, multi-miscellaneous proteins, etc., and achieves easy construction and simple expression method. , the effect of simple separation and purification process

Inactive Publication Date: 2010-01-27
INST OF BIOPHARM OF SHANDONG PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hyaluronidase currently on the market is mainly extracted from animal tissues, and its composition is relatively complex, containing more miscellaneous proteins, and it is easy to produce immune reactions to patients

Method used

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  • Hyaluronidase expression vector and application thereof
  • Hyaluronidase expression vector and application thereof
  • Hyaluronidase expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of recombinant hyaluronidase HYL

[0037] 1. Cloning of HYL gene encoding Streptococcus zooepidemicus hyaluronidase

[0038] A. Primer Design and Synthesis

[0039] According to the amino acid sequences of Streptococcus agalactiae and Streptococcus pyogenes HA lyase and the genome of Streptococcus equi, two primers were designed and synthesized, the sequences of which are as follows:

[0040] HYL1-F[5'-CGGGATCCATTTAGAGGCTGTGTGCACC-3']

[0041] HYL1-R[5'-ATACTGCAGAATGTGCTTGAATGGCTATG-3']

[0042] B. PCR reaction

[0043] Denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 5 minutes, 30 cycles; extension at 72°C for 10 minutes, and incubation at 4°C.

[0044] C. Sequencing

[0045] The PCR product was subjected to 1% agarose gel electrophoresis detection, indicating that a DNA amplification fragment with a length of about 2700bp was obtained, and after sequencing, it was s...

Embodiment 2

[0061] Embodiment two: the preparation of HA oligosaccharide

[0062] Mix the hyaluronidase enzyme solution obtained in Example 1 with the HA solution, and react at 37° C. for 2 hours. Boil for 20min and filter. Concentrate the filtrate with a rotary evaporator, take 2ml and load it on a Bio gel-P6 column (95cm×1.5cm), add NH 4 HCO 3 (0.5mol / L) is the mobile phase, and the flow rate is 12ml / h. Collect 2ml per tube. Concentrate the sugar-containing components with a rotary evaporator, and then freeze-dry to obtain HA oligosaccharides. The component analysis figure (HPLC and mass spectrogram) of the HA oligosaccharide is shown in image 3 and Figure 4 . Depend on image 3 and Figure 4 It can be seen that a high-purity HA disaccharide is obtained, the sugar has a molecular weight of 379 and contains an unsaturated double bond. By controlling the degradation time, HA oligosaccharides with different disaccharide units can be obtained.

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Abstract

The invention relates to an expression plasmid containing streptococcus zooepidemicus hyaluronidase genes and the application thereof and aims at providing a streptococcus zooepidemicus hyaluronidase expression vector, a recombinant Escherichia coil cell, a method for generating the hyaluronidase by using the cell and a method for preparing a hyaluronic acid oligose. By using a PCR method, the expression plasmid containing coding genes of the streptococcus zooepidemicus hyaluronidase is formed; a recombinant Escherichia coil strain is obtained by transforming the expression plasmid; and the hyaluronidase is generated by expressing the engineering bacterium. The hyaluronidase expression vector is an Escherichia coil expression vector containing streptococcus zooepidemicus hyaluronidase genes. The method for expressing the zooepidemicus hyaluronidase comprises the following steps of: culturing the recombinant Escherichia coil cells; expressing the hyaluronidase by controlling the temperature or IPTG induction; and using the hyaluronidase to prepare the HA oligose. The method for preparing the hyaluronidase has the characteristics of high expression level, easily expanded production and low cost. The hyaluronidase of the invention can be applied to degrading a hyaluronic acid.

Description

technical field [0001] The present invention relates to a Streptococcus zooepidemicus hyaluronidase expression vector and its application, in particular to a Streptococcus zooepidemicus hyaluronidase expression vector and recombinant E. A hyaluronidase method and a method for preparing HA oligosaccharides by using the hyaluronidase to degrade hyaluronic acid (also known as hyaluronic acid, hereinafter referred to as HA). Background technique [0002] (1) Research status of HA oligosaccharide production [0003] HA is an acidic mucopolysaccharide formed by the repeated connection of disaccharide units composed of glucuronic acid and N-acetylglucosamine. It is widely present in the tissue interstitium of animals and the membrane of some bacteria. The distribution, chemical structure, physical and chemical properties and application of HA have been widely and deeply studied at home and abroad, and it has extensive and unique application value in medicine, cosmetics, food and o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/56C12N9/42C12N1/21C12P19/14C12R1/19
Inventor 朱希强郭学平凌沛学刘菲生举正
Owner INST OF BIOPHARM OF SHANDONG PROVINCE
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