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Construction and using method of pichia pastoris expression vector facilitating achievement of natural N end expression of protein

A construction method and vector technology, applied in the construction field of Pichia pastoris expression vector, can solve the problems such as complicated construction process

Inactive Publication Date: 2012-06-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since pPIC9K contains two Xho I restriction sites, the construction process of the expression plasmid at the natural N-terminus of the protein is cumbersome.

Method used

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  • Construction and using method of pichia pastoris expression vector facilitating achievement of natural N end expression of protein
  • Construction and using method of pichia pastoris expression vector facilitating achievement of natural N end expression of protein
  • Construction and using method of pichia pastoris expression vector facilitating achievement of natural N end expression of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Plasmid pPIC9K M build

[0033] Plasmid pPIC9K M The build process is as follows figure 2 ,Specific steps are as follows:

[0034] 1) Design a pair of forward and reverse primers KanF and KanR according to the sequence between Xho I and Sph I at the Kanna resistance gene in pPIC9K,

[0035] KanF: 5′- GTC GAC CAAGACGTTTCCC-3' (SEQ ID NO. 1);

[0036] KanR: 5′- GTGCAT GCAAGGAGATGGC-3' (SEQ ID NO. 2);

[0037] The 5' end of KanF contains a Sal I restriction site (shown underlined), and the 5' end of KanR contains a Sph I restriction site (shown underlined). Under the action of KanF and KanR, the KanXS fragment was amplified by PCR using the pPIC9K plasmid as a template (94°C for 3min; 30 cycles, 94°C for 30s, 55°C for 30s, 72°C for 30s; 72°C for 10min). The PCR product was analyzed by 0.7% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-KanXS), transformed into Escherichia coli JM109, and sent to Shanghai Sang...

Embodiment 2

[0039] Example 2 Construction and heterologous expression of the natural N-terminal expression plasmid of the 10th family xylanase of Aspergillus usami

[0040]1) Synthetic primer XynCF2: 5'- CTCGAG AAAAGACAGGCTTCAGTGAGTATTGA-3', XynCR2:5'- GCGGCCGC CTAGAGAGCATTTGCGATAG-3′, underlined are Xho I and Not I restriction sites respectively. Using the vector pUCm-T-Ausxyn10A plasmid as a template, the xylanase gene Ausxyn10A was obtained by PCR (94°C for 3min; 30 cycles, 94°C for 30s, 56°C for 30s, 72°C for 1min; 72°C for 10min). After agarose gel electrophoresis analysis, the target band was recovered and ligated with pUCm-T (pUCm-T-Ausxyn10A'), transformed into Escherichia coli JM109, and sent to Shanghai Sangon for sequencing after being identified by enzyme digestion.

[0041] 2) Combine the sequenced correct pUCm-T-Ausxyn10A' with pPIC9K M Plasmids were double digested with Xho I and Not I, and the recovered digested products were ligated under the action of T4DNA ligase t...

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Abstract

The invention relates to a construction and using method of a pichia pastoris expression vector facilitating achievement of natural N end expression of protein. The expression vector is formed by modifying a vector pPIC9K based on an isocaudarner principle only contains a single Xho I enzyme cutting site, and a pPIC9KM structure is shown in graph 1. After a target gene is connected to a Kex2 enzyme cutting site sequence in a pPIC9KM plasmid through a polymerase chain reaction (PCR) and a double-enzyme-cutting process, the target gene is secreted to the outside of a cell under the guidance of a secretion signal, signal peptide is processed and removed through Kex2 during the process, and the protein of the natural N end is achieved.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a construction method and application demonstration of a Pichia pastoris expression vector that facilitates the expression of the natural N-terminus of the protein. Background technique [0002] With the rapid development of molecular biology and genetic engineering technology, foreign proteins have been successfully expressed in Escherichia coli, insect cells and even mammalian cells. Although these expression systems have been widely studied and applied, there are still There are many shortcomings and deficiencies. In contrast, Pichia pastoris has a shorter growth cycle, simple genetic manipulation, and has a eukaryotic protein synthesis pathway, which can carry out eukaryotic modification of proteins (protein processing, folding, post-translational modification, etc.). At the same time, Pichia pastoris can High-density growth on simple medium, even in large-scale ferme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66
Inventor 邬敏辰汪俊卿陈伟李剑芳张慧敏唐存多
Owner JIANGNAN UNIV
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