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Candida utilis expression vector and construction method thereof

A technology for producing Candida utilis and an expression vector, which is applied in the fields of expressing exogenous proteins and molecular biology to save human, material, and financial resources.

Inactive Publication Date: 2009-07-22
THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the expression vector of Candida utilis has not been commercialized, so it will be of great significance to explore a feasible construction method of expression vector of Candida utilis or construct a commercial expression vector of Candida utilis

Method used

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  • Candida utilis expression vector and construction method thereof
  • Candida utilis expression vector and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Primer design

[0019] According to the gene sequence of C.utilis published in GenBank combined with pBR322 plasmid sequence characteristics, primers were designed and suitable restriction sites were introduced. Among them, primers P5 and R5 were designed according to literature. The primers are as follows:

[0020] P1: 5’-GGATATCTTACAGCGAGCACTCAA-3’ (Introduction of Eco RV restriction site)

[0021] R1: 5'-AGGTACCGCTAGCTCTAGAATGTTGTTTGT-3' (introduction of Kpn I, Nhe I and Xba I restriction sites)

[0022] P2: 5’-ttctagagctagcggtacctatgacttttat-3’ (introduction of Xba I, Nhe I and Kpn I restriction sites)

[0023] R2: 5'-gggatccACGTGTAATACCAGG-3' (introduction of Bam HI restriction site)

[0024] P3: 5’-cgatatcTGCCAGTAGTCATATGC-3’

[0025] R3: 5’-cgatatcTGACTTGCGCTTACTAG-3’ (Introduce Eco RV restriction sites at the 5’ ends of both primers)

[0026] P4: 5'-CgtcgacAGTAAGTATGAAAAGAGC-3' (Introduction of Sal I site)

[0027] R4: 5’-GggatccGG GTTTGGTCTATGTTGCT-3’ (i...

Embodiment 2

[0032] Example 2: Construction of homologous integration expression vector pGLR9K

[0033] The C.utilis genome was used as a template and P4 / R5 was used as primers to perform PCR to obtain upstream fragments of the L41 gene. Similarly, the C.utilis genome was used as a template and P5 / R4 as primers to perform PCR to obtain downstream fragments of the L41 gene. The two fragments were recovered separately, and the two fragments were mixed in equal amounts as templates, and PCR was performed with P4 / R4 as primers to obtain the CYH resistance gene. The PCR reaction conditions were: 94°C for 5 min; 94°C denaturation for 30 seconds, 56°C annealing for 30 seconds, 72°C extension for 2 minutes, 30 cycles, 72°C extension for 10 minutes. The recovered target fragment was connected to the vector pMD19-T simplevector (named pT-CYH) for sequencing analysis.

[0034] Use C.utilis genome as template and P1 / R1 as primers to perform PCR to obtain yeast GAP promoter (GAP-p) gene fragment; also use ...

Embodiment 3

[0039] Example 3: Construction of recombinant expression vector containing target gene

[0040] Using plasmid pUC19-XA as a template and P6 / R6 as primers, PCR was performed to obtain the xynA target gene fragment. The PCR conditions were: 94°C for 5 min; 94°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1.5 minutes, 30 cycles; 72°C extension for 10 minutes. The target gene was recovered and cloned into the vector pMD19-T simple vector (named pT-XA) for sequencing analysis.

[0041] The pT-XA plasmid was extracted, digested with Xba I / Kpn I, and the target fragment was recovered and constructed into the vector pGLR9K multiple cloning site to obtain the plasmid pGLXR. Transform E. coli DH5α, screen and identify positive clones and save them for later use. See attached figure 2

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Abstract

The invention discloses an expression carrier of an industrial microorganism that is candida utili and simultaneously provides a construction method of the candida utili expression carrier. The invention uses a basic skeleton of a pBR322 carrier and a template of candida utili genomic DNA, obtains a cycloheximide resistance gene with the site-directed mutagenesis method, obtains a rDNA segment and a GAP promoter-terminator segment of the candida utili genomic DNA by adopting the PCR method and the template of the candida utili genomic DNA, and constructs the integrated candida utili expression carrier that is mediated by 18S rDNA and has cycloheximide resistance. The integrated candida utili expression carrier can be used for preparing a foreign candida utili expression protein and can be widely applied into the fields of feedstuff addition agents, food industry, and pharmacy, and the like.

Description

Invention field [0001] The present invention relates to the field of molecular biology, in particular to the establishment of a transformation system of Candida utilis for the technical field of expressing foreign proteins. Background technique [0002] The E. coli expression system is by far the most effective and convenient expression system, but the large amount of foreign protein expression in E. coli is in the form of insoluble inclusion bodies in the cell. As an expression host, E. coli cannot perform specific post-translational modifications, especially the expression of eukaryotic genes, which may cause the product to lose biological activity. In order to overcome the shortcomings of the E. coli expression system, a new eukaryotic expression system needs to be developed. The yeast expression system has post-transcriptional processing and modification functions, simple operation, low cost, suitable for stable expression of functional foreign proteins, and large-scale ferme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/56
Inventor 王炜包慧芳崔卫东詹发强杨红兰
Owner THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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