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
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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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