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Method for constructing tandem expression vector and engineering strain of Pinctada martensii muscle antioxidant peptide

A technology of pearl oyster and antioxidant peptides in Hepu, applied in the field of genetic engineering, can solve the problems of unsuitability, increased synthesis cost, wrong protein, etc., and achieve the effects of low price, high purity and simple culture conditions

Inactive Publication Date: 2016-02-17
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Gene recombination technology is a relatively popular method in the world at present, and it is one of the research hotspots for the preparation of a large number of polypeptides. In the prior art, there are the following three in vitro gene tandem methods: one is random directional tandem method, and the result of this method is non-directional It is random, which may lead to wrong proteins during the expression and translation process; the second is the chemical splicing method, which is not suitable for constructing multiple copies (more than 4 times), because the cost of artificially synthesized sequences is high; The third is the PCR amplification tandem method, which is similar to the chemical splicing method. As the copy number increases, the synthesis cost increases

Method used

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  • Method for constructing tandem expression vector and engineering strain of Pinctada martensii muscle antioxidant peptide
  • Method for constructing tandem expression vector and engineering strain of Pinctada martensii muscle antioxidant peptide
  • Method for constructing tandem expression vector and engineering strain of Pinctada martensii muscle antioxidant peptide

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Embodiment 1

[0030] The construction method of the tandem expression vector and the engineering strain of the Hepu pinnacle oyster meat antioxidant peptide (PFMAP) involved in the present invention is as follows:

[0031] 1. Modification of the haploid gene sequence of Hepu pinnacle oyster meat antioxidant peptide (PFMAP)

[0032] According to the codon preference of the Pichia genetic engineering strain, use the computer software PrimerPremier5.0 to design the 10 amino acid sequences (Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg, see Patent application number 201410040473.4) Corresponding gene sequence: 5'-GGTGCTGGTTTGCCAGGTAAGAGAGAGAGA-3'; AT content is 0.46, GC content is 0.54. The enzyme cleavage sites added at both ends of the active peptide are restriction endonucleases XhoI and SalI respectively; in order to cut the enzyme cleavage sites smoothly by the restriction endonucleases in the later stage, choose to add 3 to 5 protection bases (three protection bases of CCG are added to the lef...

Embodiment 2

[0067] The construction method of the tandem expression vector and engineering strain of the Hepu pinnacle oyster meat antioxidant peptide (PFMAP) provided in this example is as follows:

[0068] The prokaryotic vectors for modifying the haploid gene sequence of Hepu pinnacle oyster meat antioxidant peptide (PFMAP) and constructing the haploid are the same as in Example 1.

[0069] 1. Construction of KS-2PFMAP, KS-4PFMAP, KS-6PFMAP, KS-12PFMAP recombinant vectors

[0070] The construction methods of KS-2PFMAP, KS-4PFMAP, KS-6PFMAP and KS-12PFMAP recombinant vectors are the same. The following uses KS-2PFMAP as an example to illustrate the operation method:

[0071] 2. Construction of KS-2PFMAP recombinant vector

[0072] (1) Single enzyme digestion and dephosphorylation of KS-PFMAP

[0073] The recombinant vector KS-PFMAP obtained in Example 1 was digested with the restriction endonuclease QuickXhoI according to the 20 μL digestion system in a metal bath at 37°C for 1 h, and...

Embodiment 3

[0099] The difference from Example 2 is that the construction method of KS-4PFMAP is as follows: use the constructed pBluescriptⅡKS-2PFMAP as a template, use the general primers M13+, M13- to carry out PCR reaction to obtain the fragment gene containing the target band of 2PFMAP, and then use it to restrict The recombinant vector pBluescriptⅡKS-2PFMAP can be obtained by double digestion with endonuclease XhoI and SalI and connected with the recombinant vector pBluescriptⅡKS-2PFMAP after single digestion and dephosphorylation. Similarly, KS-6PFMAP or KS-6PFMAP can be obtained. KS-12PFMAP recombinant vector.

[0100]With reference to the same method in Example 2, Pichia engineering strain GS115-pPICZαA-pBluescriptⅡKS-4PFMAP, Pichia engineering strain GS115-pPICZαA-pBluescriptⅡKS-6PFMAP or Pichia engineering strain GS115-pPICZαA-pBluescriptⅡKS-12PFMAP can be obtained . Prepared engineered strains such as Figure 2-4 shown in .

[0101] Recombinant vectors KS-PFMAP, KS-2PFMAP, ...

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Abstract

The invention discloses a method of utilizing methods of molecular biology and gene engineering to construct a polyploid tandem gene pBluescriptIIKS-nPFWM, a Pichia pastoris tandem expression vector pPICZ alpha A-pBluescriptIIKS-nPFWM and a Pichia pastoris engineering strain GS115-pPICZ alpha A-pBluescriptIIKS-nPFWM1, wherein n is 1, 2, 4, 6 or 12. A foundation is laid for subsequent efficient and stable expression of antioxidant peptide, industrialized large-scale production and application in the field of cosmetics and healthcare food.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tandem expression vector of Hepu pearl oyster meat antioxidant peptide and a method for constructing an engineering strain. Background technique [0002] Hepu pearl oyster, also known as Martensii pearl oyster, belongs to the mollusc phylum bivalve pearl oyster order pearl oyster family. It is the main species of marine cultured pearl oyster and produces the mother oyster of "South Pearl". It is mainly distributed in Guangxi, Guangdong and the southern coastal areas of the Taiwan Strait. It is one of the artificially cultured seawater pearl shellfish with the widest breeding area and the largest number in my country. Hepu pearl oyster meat is a high-quality protein source seafood with high protein, low fat, low sugar and high nutritional value. Its crude protein dry weight content is between 66.71% and 81.2%, which has high development and utilization va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/81C12N1/19C12R1/84
Inventor 吴燕燕马永凯李来好杨贤庆赵永强王锦旭林婉玲杨少玲邓建朝马海霞魏涯
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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