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Plasmid, recombinant engineering bacteria and method for preparing hyaluronic acid with uniform molecular weight

A technology of recombinant engineering bacteria and hyaluronic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve problems such as unrealization

Active Publication Date: 2016-12-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above technology can realize the heterologous synthesis of hyaluronic acid in Bacillus subtilis, if more refined hyaluronic acid, that is, homogeneous molecular weight hyaluronic acid, is efficiently prepared, and homogeneous hyaluronic acid with different molecular weights is obtained through artificial regulation, now Not yet possible with technology

Method used

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  • Plasmid, recombinant engineering bacteria and method for preparing hyaluronic acid with uniform molecular weight
  • Plasmid, recombinant engineering bacteria and method for preparing hyaluronic acid with uniform molecular weight
  • Plasmid, recombinant engineering bacteria and method for preparing hyaluronic acid with uniform molecular weight

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Cloning of hyaluronan synthase gene and construction of integrated expression vector

[0086] (1) Cloning of Pasteurella multocida Hyaluronate synthase gene

[0087] Genomic DNA of Pasteurella multocida (Pasteurella multocida P-1059ATCC#15742) was extracted using the Tiangen Bacteria Genomic DNA Extraction Kit, and a pair of primers were designed to perform PCR reaction using the extracted Genomic DNA of Pasteurella multocida as a template , the specific reaction conditions were pre-denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 1 minute, 48°C for 1 minute, and 72°C for 2 minutes and 30 seconds, and finally 72°C for 8 minutes. The sequence of a pair of primers designed is as follows:

[0088] 5'-CGACTAGTATGAATACATTATCACAAGCAATAAAAGC-3' (with a Spe I restriction site at the 5' end, ACTAGT), primer SEQ NO.12.

[0089]5'-CGGGATCCCTAAATATCTTTTAAGATATCAATCT-3' (with a BamH I restriction site at the 5' end, GGATCC), primer SEQ NO.13.

[00...

Embodiment 2

[0093] Example 2: Cloning of enzyme genes related to hyaluronic acid precursor synthesis and construction of episomal stringent expression vectors

[0094] (1) Cloning from Bacillus subtilis UDP-glucose dehydrogenase gene (UDP-Glucose dehydrogenasegene)

[0095] Genomic DNA of Bacillus subtilis (Bacillus subtilis subsp.subtilis168) was extracted using the Tiangen Bacteria Genomic DNA Extraction Kit, a pair of primers were designed, and the extracted Bacillus subtilis genomic DNA was used as a template for PCR reaction. The specific reaction conditions were pre-denaturation at 94°C for 5 Minutes, followed by 30 cycles at 94°C for 1 minute, 48°C for 1 minute, and 72°C for 1 minute and 30 seconds, and finally according to the program of 72°C for 8 minutes. The sequence of a pair of primers designed is as follows:

[0096] 5'-CGGGATCCGGAGAGGGTTGAGCGCTGTGAA-3' (with a BamH I restriction site at the 5' end, GGATCC), primer SEQ NO.14.

[0097] 5'-GCTCTAGATTATAAATTGACGCTTCCCAAGTCTTT...

Embodiment 3

[0120] Example 3: The expression vectors constructed in Examples 1 and 2 were respectively transformed into Bacillus subtilis host cells to obtain recombinant engineered strains, and several homogeneous hyaluronic acids with different molecular weights were prepared by fermentation.

[0121] This example describes the transformation of the expression vectors constructed in Examples 1 and 2 into Bacillus subtilis host cells by preparing competent methods to obtain recombinant engineering strains, and prepare several homogeneous hyaluronic acids with different molecular weights by fermentation .

[0122] (1) The constructed integrated expression vector pAX01-PmHAS was transformed into Bacillus subtilis cells to obtain the recombinant engineering strain B. subtilis (pAX01-PmHAS).

[0123] Prepare the competent state of Bacillus subtilis according to the following method: Streak Bacillus subtilis on the LB plate to obtain a single colony, then inoculate the single colony into 3mL li...

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Abstract

A plasmid, recombinant engineering bacteria and a preparation method of hyaluronic acid with uniform molecular weight belong to the technical field of gene engineering. A first expression plasmid containing a Pasteurella multocida hyaluronan synthase gene (SEQ NO.1 in the sequence table) and a second recombinant expression plasmid containing hyaluronic acid precursor synthesis related genes (SEQ NO.2, SEQ NO.3, SEQ NO.4 and SEQ NO.5 in the sequence table) cloned from Bacillus subtilis are introduced into gram positive safe microorganisms, and an expression regulation and control system consisting of the two recombinant expression plasmids respectively controls hyaluronan synthase and hyaluronic acid precursor synthesis related enzyme, so as to obtain hyaluronic acid with uniform molecular weight by a biosynthesis method. The method provided by the invention not only improves the capability of the host strains to synthesize hyaluronic acid, but also can obtain several varieties of sufficient pure hyaluronic acid with uniform molecular weight.

Description

technical field [0001] The invention relates to a method in the technical field of genetic engineering of microorganisms, in particular to a biosynthetic method for preparing several hyaluronic acids with uniform molecular weights for safe engineering strains by using Gram-positive safe microorganisms constructed in the form of plasmids and genome integration. Background technique [0002] Hyaluronic acid (Hyaluronic Acid, Hyaluronan, HA) is a linear unbranched macromolecule formed by repeated cross-linking of disaccharide units composed of β-D-glucuronic acid (GlcUA) and β-D-N-acetylglucosamine (GlcNAc). Acid mucopolysaccharides. The average molecular weight (MW) range of natural hyaluronic acid is 2×10 4 -7×10 7 Da. HA with different molecular weights has different biological functions: high molecular weight HA (HMW-HA, MW>2×10 6 ) has high viscoelasticity, plasticity, permeability and good biocompatibility. Clinically, hyaluronic acid is mainly injected into the kn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N15/74C12N1/21C12P19/26C12R1/125C12R1/10C12R1/11C12R1/085C12R1/08C12R1/07C12R1/01
Inventor 高向东姚文兵贾毓宁陈晓菲
Owner CHINA PHARM UNIV
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