Recombined endo-chitinase gene order and recombinant vector threreof

A technology of chitinase gene and recombinant vector, which is applied in the field of genetic engineering, can solve the problems of difficulty in controlling the degree of degradation, high energy consumption of acid degradation, secondary pollution of the environment, etc., and achieve efficient and cheap industrial production, easy purification, The effect of broad application prospects

Inactive Publication Date: 2008-11-19
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Acid degradation consumes a lot of energy, and the degree of degradation is difficult to control. The degradation products are mainly monosaccharides. The waste water containing sulfuric acid or hydrochloric acid generated during the degradation process is discharged directly or neutralized with alkali and then discharged into the environment, causing serious secondary damage to the environment. Pollution

Method used

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  • Recombined endo-chitinase gene order and recombinant vector threreof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Example 1. Acquisition of recombinant endochitinase gene sequence based on Pichia pastoris codon bias

[0011] According to the codon preference of Pichia pastoris and the gene sequence of endochitinase, the following primers were designed:

[0012] f 1 : 5'-GAATTCGCTAGTGGTTACGCTAACGCTGTTTACTTTACTAACTGGGGTATTTACGGT-3'

[0013] f 2 : 5'-ACTGGGGTATTTACGGTCGTAACTTTCAACCACAAAACCTTGTTGCTTCTGATATTACT-3'

[0014] f 3 : 5'-TGTTGCTTCTGATATTACTCATGTTATTTACTCTTTTATGAACTTTCAAGCTGATGGTACT-3'

[0015] f 4 : 5'-TTCAAGCTGATGGTACTGTTGTTTCTGGTGATGCTTACGCTGATTACCAAAAGCATTAC-3'

[0016] f 5 : 5'-ATTACCAAAAGCATTACGATGATGATTCTTGGAACGATGTTGGTAACAACGCTTACGGT-3'

[0017] f 6 : 5'-GTAACAACGCTTACGGTTGTGTTAAGCAACTTTTTTAAGTTGAAGAAGGCTAACCGTAAC-3'

[0018] f 7 : 5'-AGAAGGCTAACCGTAACTTGAAGGTTATGCTTTCTATTGGTGGTTGGACTTGGTCTACT-3'

[0019] f 8 : 5'-GTTGGACTTGGTCTACTAACTTTCCATCTGCTGCTAGTACTGATGCTAACCGTAAGAAC-3'

[0020] f 9 : 5'-ATGCTAACCGTAAGAACTTTGCTAAGACTGCTATTACTTTTTATGAAGGATTGGGGTTTT-...

Embodiment 2

[0044] Example 2. Construction of recombinant Pichia pastoris expression vector

[0045] The PCR amplification product and the original expression plasmid pPIC9K were double-digested with EcoRI and NotI respectively, and the target band was recovered by agarose gel electrophoresis. 4 DNA ligase ligated the recovered target bands to obtain the eukaryotic expression vector pSECH, and used CaCl 2 Transform it into Escherichia coli DH5α by heat shock at 42°C for 90 seconds. Single colonies were screened for Kana resistance. Enzyme digestion identification and DNA sequencing of the selected transformed clones prove that the clones are correct and then sent to Shanghai Bioengineering Co., Ltd. for sequencing. For the specific operation process, see figure 1 .

Embodiment 3

[0046] Example 3. Transformation and screening of recombinant Pichia pastoris

[0047] The identified recombinant expression vector pSECH plasmid DNA was linearized with endonuclease XbaI, and transformed into Pichia pastoris GS115 by electroporation. Immediately after the electric shock, 1ml of pre-cooled 1mol / l sorbitol was added, centrifuged at 3000rpm for 5min, the bacteria were resuspended in 400μl of pre-cooled 1mol / l sorbitol, and 200μl was spread on MD plates (1.34% yeast nitrogen base, 4 ×10 -5 % biotin, 2% glucose, 2% agarose), cultured at 30°C until colonies appeared. Randomly pick bacterial colonies and plant them with different concentrations of antibiotics G418 (0, 0.25mg / ml, 0.50mg / ml, 0.75mg / ml, 1.00mg / ml, 1.50mg / ml, 1.75mg / ml, 2.00mg / ml, 3.00mg / ml, 4.00mg / ml) YPD plate, cultured at 30°C for 2-5 days, and checked the growth of the colony every day. According to the growth situation, the positive clone transformants with high G418 resistance were quickly scre...

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Abstract

The invention provides a recombined endo-chitinase gene sequence based on Pichia pastoris codon preference and a recombined vector containing the recombined gene. The invention adopts modern biotechnology and makes use of PCT so as to synthesize the recombined endo-chitinase gene sequence based on Pichia pastoris codon preference, and then clones the gene sequence in a vector pPIC9K so as to obtain a eukaryon expression vector pSECH. The transformation of Pichia pastoris GS115 is carried out through adopting an electric shock method, and quick screening of positive cloning transformant with higher resistance is carried out according to growth conditions, thereby realizing efficient industrialized production of endo-chitinase at low cost. Moreover, the recombined endo-chitinase gene sequence and the recombined vector have wide application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant endochitinase gene sequence based on the codon preference of Pichia pastoris and a recombinant vector containing the recombinant endochitinase gene. Background technique [0002] Chitin, also known as chitin, has a chemical name of (1,4)-2-acetylamino-2-deoxy-β-D-glucose, which is formed by connecting N-acetylglucosamine through β-1,4-glycosidic bonds. The natural macromolecular aminopolysaccharide compounds are widely present in the cells of lower plant fungi, algae and crustacean shrimp, crab, insect shells and cell walls of higher plants. The annual biosynthesis of chitin in nature is nearly 10 billion tons, of which 10% comes from the ocean. It is the second largest renewable resource after cellulose on the earth, but unfortunately this resource has not been fully utilized. The natural loss of waste has not only caused huge waste, but also caused serious envi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/63C12N1/19C12R1/84
Inventor 于平励建荣唐云平
Owner ZHEJIANG GONGSHANG UNIVERSITY
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