Hyaluronidase encoding gene and fermentation production and purification method thereof

A hyaluronidase and gene technology, applied in the field of bioengineering, can solve the problems of extraction and separation steps limiting the wide application of leech hyaluronidase

Active Publication Date: 2014-04-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sources of leech raw materials and the cumbersome extraction and separation steps have greatly limited the wide application of leech hyaluronidase in medical and scientific research

Method used

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  • Hyaluronidase encoding gene and fermentation production and purification method thereof
  • Hyaluronidase encoding gene and fermentation production and purification method thereof
  • Hyaluronidase encoding gene and fermentation production and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Wild leech RNA is extracted

[0026] Take a living leech, cut about 100 mg of head tissue, and freeze it quickly with liquid nitrogen. The tissue was fully ground by liquid nitrogen grinding method, and the total RNA extraction kit from tissue / cell (Hangzhou Baosai Biotechnology Co., Ltd.) was used for extraction. Add 1ml of lysate, vortex and mix quickly to fully lyse the cells and inactivate RNase, and incubate at room temperature for 2 minutes. Add 0.2ml of chloroform, shake and mix vigorously to make the sample turn milky white, and let it stand for 2-3min. Centrifuge at 12,000 rpm at 4°C for 10 min, divide the sample into three layers, and transfer the RNA-containing aqueous phase in the upper layer to a new tube. Add 1 / 2 volume of absolute ethanol, invert and mix well, transfer all to the adsorption column, and centrifuge at 12,000 rpm at 4°C for 1 min. Discard the filtrate, add 500 μl protein-removing solution, centrifuge at 12,000 rpm at 4°C for...

Embodiment 2

[0027] Example 2: Preparation of cDNA by reverse transcription

[0028]The reverse transcription cDNA was prepared using M-MLV First Strand RT kit (Hangzhou Baosai Biotechnology Co., Ltd.). Using the extracted leech RNA as a template, configure the reaction mixture according to the following (20 μl) system: 5×First-Strand buffer, 4 μl; Oligo(dT) 18 Primer (50 μM), 1 μl; 10 mM dNTP, 1 μl; RNase Inhibitor (40 U / μl), 1 μl; M-MLV (200 U / μl), 1 μl; RNA, 12 μl. The reaction system was placed in a PCR instrument at 50°C for 1 h. The reaction was terminated by heating at 70°C for 10 min. Store on ice for subsequent experiments or store at -80°C.

Embodiment 3

[0029] Embodiment 3: Acquisition of leech hyaluronidase gene

[0030] By searching and analyzing the leech EST in the Expressed sequence tags (EST) of the NCBI database, two incomplete mRNA genes (GenBank: JZ186329.1 and GenBank: FP652258.1) were found to be similar to the heparanase gene (homology lower than 40%), and by further comparison with other hyaluronidases, it was preliminarily confirmed that these two incomplete mRNA sequences were leech hyaluronidase sequences. According to these two sequences, two degenerate primers (EST1: CTGGTGMYCACRTAACYGCTTTTAC; EST2: TCAACATACCTTGAYGCYWCWTA) were designed near the 5-end. The degenerate primers are respectively used as upstream primers, and the downstream primers are Oligo(dT) 18 , using the cDNA prepared in Example 2 as a template, PCR amplifies the target fragment. Amplified by PCR, obtained with primers EST1 and Oligo(dT) 18 Amplified target fragment of about 900bp. The target fragment was recovered, connected to the PM...

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Abstract

The invention discloses a hyaluronidase gene having a nucleotide sequence as shown in (a) or (b) or (c), wherein the nucleotide sequence (a) is as shown in SEQ ID NO.1; the nucleotide sequence (b) is obtained by substituting, deleting or adding one or more nucleotides on the basis of the nucleotide sequence (a) and has hyaluronidase activity; and the nucleotide sequence (c) has the whole similarity of over 85% with an amino acid sequence (a) and has hyaluronidase activity. According to the invention, the efficient heterologous expression and purification of the obtained hyaluronidase from leeches is successfully realized by using a pichia pastoris expression system. A leech hyaluronidase product prepared through recombination belongs to a pharmacological active compound and can be used in the field of medicines such as drug dispersing agents and the like for treating cardiovascular and cerebrovascular diseases, ophthalmic diseases and tumor. The hyaluronidase gene lays the foundation for the industrial preparation of the hyaluronidase from the leeches.

Description

technical field [0001] The invention relates to a hyaluronidase gene and its fermentation production and purification method, in particular to a hyaluronidase gene derived from leeches, belonging to the technical field of bioengineering. Background technique [0002] Hyaluronidase was discovered by Duran Reynals in 1928 and called diffusion factor, and was officially named hyaluronidase (HAase) by Chain and Duthie in 1940. Hyaluronidase is widely found in eukaryotes and prokaryotes, and it mainly hydrolyzes hyaluronic acid, which is an important physiologically active substance. Hyaluronidases are classified into three classes according to their substrate specificity and catalytic mechanism of action. The first category is the hyaluronic acid 4-glycosyl hydrolase family derived from mammals. This type of hyaluronidase (EC3.2.1.35) is a glycosidase with hydrolysis and transglycosidic activity. HA) β-1, 4-glycosidic bond, the product is mainly tetrasaccharide hyaluronic acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N1/19
Inventor 陈坚堵国成康振金鹏
Owner JIANGNAN UNIV
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