Transgenic vector and transgenic pig for salivary gland tissue-specific expression of exogenous protein and construction method thereof

A technology of tissue-specific, transgenic vectors, applied in the field of genetic engineering

Active Publication Date: 2016-04-06
国科润风(广州)生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the gene is mainly regulated by the upstream regulatory region (11.5Kb) and downstream sequence (about 2.5-3kb) of the parotid protein gene (i.e., the parotid protein promoter PSP), but its regulatory gene is only regulated and expressed in the salivary gland. Other tissues and organs in the whole body do not express

Method used

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  • Transgenic vector and transgenic pig for salivary gland tissue-specific expression of exogenous protein and construction method thereof
  • Transgenic vector and transgenic pig for salivary gland tissue-specific expression of exogenous protein and construction method thereof
  • Transgenic vector and transgenic pig for salivary gland tissue-specific expression of exogenous protein and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of the transgene expression vector pPB-MusPSP-neo-EGFP-BgEgXyAp

[0072] Include the following steps:

[0073] 1. Obtaining the target gene

[0074] According to literature reports, a series of acid-resistant glucanase genes (cel4T (alicyclic acid bacillus), beta-1, 3(4)-glucanase (Paecilomyces sp.), Bg17A from Bisporasp.MEY-1, eg1314 from Bacillus licheniformis, C-APPA from Citrobacterfreundii, AppA from Escherichia coli, and XYNB from Aspergillus niger, remove the corresponding signal peptides from these genes Sequence, plus the signal peptide mature peptide sequence of porcine parotid gland protein, and artificially synthesized after optimization according to porcine codons. Then express in porcine kidney PK15 cells in mammalian expression system, and screen for genes with high enzyme activity secreted in porcine cells , the present invention screens out the optimized glucanase genes Bg17 (SEQ ID NO: 1) and Eg1314 (SEQ ID NO: 2) and xylanase ...

Embodiment 2

[0141] Example 2 Using the transgenic vector constructed in Example 1 to construct a transgenic pig

[0142] 1. Screening of transgenic cells

[0143] Using the BXT electroporation instrument, the constructed pPB-MusPSP-neo-EGFP-BgEgXyAp vector and the transposase plasmid pCMV-mPB (a gift from the Wellcome Trust Sanger Institute, the applicant has removed the neo gene) were transfected into pig fetuses at a molar ratio of 1:1 Fibroblasts (boar), transfection conditions: 310V, pulse time 1ms, pulse times 3 times. The cells after electrotransfection were plated, and after 36 hours, they were subcultured according to the ratio of 1:6. Cell Selection Medium Preparation Method 42.5% GlutaMAX TM (Life Company) + 42.5% high sugar DMEM (Life Company) + 15% FBS, first add G418 300ug / ml to the cell selection medium, and after 5 days of cell selection after electrotransfection, use 200ug / ml concentration to continue screening After 10 days, change to 80ug / ml to maintain screening, and...

Embodiment 3 Embodiment 2

[0160] Example 3 Transgenic expression effect detection of the transgenic pigs constructed in Example 2

[0161] After two months of age, the transgene expression effect of the transgenic pigs constructed in Example 2 was detected, mainly by detecting glucanase (β-glucannase) in the saliva of the transgenic pigs (wherein No. 707 pigs), xylem Carbohydrase (Xylanase), phytase (phytase) enzyme activity. The detection method is: use a sponge or rubber rod to let the piglet bite, collect 50ul of piglet saliva, and measure it in acetic acid-sodium acetate buffer solution (HAC-NaAC) at 39.5°C. Three technical repetitions were performed, and the saliva of three non-transgenic pigs of the same age in the same house was mixed as a control (wild type). Detect the activity of recombinant β-glucanase, xylanase and phytase. Mean ± S.D. was used for statistical analysis. The enzyme activity assay results were as Figure 18 shown, from Figure 18 It can be seen that the β-glucanase (β-gl...

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Abstract

The invention discloses a transgenic vector of salivary gland tissue specific expression foreign protein and a construction method thereof; the expression vector is constructed by inserting a recombinant fusion gene constructed by glucanase gene, xylanase gene, and phytase gene into a parotid gland-specific transgenic vector; the vector is introduced into pig genome, and transgenic pigs is produced by a somatic cell nucleus transplant technique. The transgenic vector of the invention has hydrolytic action on glucanase in barley raw materials, xylanase in wheat and corn, and phytase in vegetable feed; the pig salivary gland is used as a reactor to realize secretion for the whole life; the addition of the enzyme preparations into feed is replaced permanently, which gives better play to the enzymes; the expression vector of the invention has a piggyback transposon, which greatly increases the transgenosis efficiency, changes the transgenic integration model of common plasmid serial recombination into single locus single copy integration, and thus better simulates the internal environment of biological genes.

Description

technical field [0001] The technology of the invention belongs to the field of genetic engineering, and more specifically, the invention relates to a transgenic vector for salivary gland tissue-specific expression of foreign proteins, a transgenic pig and a construction method thereof. Background technique [0002] β-glucan belongs to the structural non-starch polysaccharide in the plant cell wall. It is a D-glucose polymer connected by β-1,3 and β-1,4 glycosidic bonds. Its molecular weight is about 6500KDa and above. Its solubility is affected by the content of glycosidic bonds in the structure and the degree of polymerization. It is widely present in the cell walls of higher plants, and has a high content in the endosperm cell walls of barley, wheat, rye and other cereal crops. In barley, β - Dextran content about 4%-10%. β-glucanase (β-glucanase) can hydrolyze β-glucan in grains such as barley, wheat and rye, and catalyze the cleavage of β-1,3 and β-1,4 glycosides in β-g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/85A01K67/027
Inventor 吴珍芳张献伟李紫聪刘德武贺晓燕蔡更元郑恩琴
Owner 国科润风(广州)生物医药有限责任公司
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