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Method for overexpression of cholate activation lipase in mammary gland of mammal

A mammalian, overexpression technology, applied in the field of genetic engineering, can solve problems such as uncertain effects

Inactive Publication Date: 2013-05-08
BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the role of these cis-elements in regulating the function of BSSL gene expression has not been determined

Method used

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  • Method for overexpression of cholate activation lipase in mammary gland of mammal
  • Method for overexpression of cholate activation lipase in mammary gland of mammal
  • Method for overexpression of cholate activation lipase in mammary gland of mammal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of human BSSL breast expression vector

[0029] Using IRATp970H1068D plasmid as a template, the cDNA sequence of human BSSL gene was amplified by PCR. A pair of primers used to amplify the cDNA sequence are hBSSL-F (5'-CCGCTCGAGACCATGCTCACCATGGGGCGC-3') and hBSSL-R (5'-CCGCTCGAGTTCAGGGGTATGAGGCTTTAT-3'), respectively introducing XhoI restriction sites in the primers. The amplified sequence was recovered and cloned into the pBC1 vector digested with XhoI ( figure 1 ), the sequence and direction of the inserted fragment were identified by PCR, and sequenced, and finally the correct expression vector inserted was named pBC-hBSSL.

Embodiment 2

[0030] Example 2 Establishment and detection of transgenic mouse model

[0031] 1.1 Production of transgenic mice by microinjection

[0032] Studies have shown that in the production of transgenic mice by microinjection, linear DNA has a higher integration efficiency than circular DNA. Therefore, the constructed expression vector pBC-hLPL should be digested to remove useless prokaryotic parts and linearized before microinjection.

[0033] The constructed expression vector pBC-hBSSL was digested with NotI / SalI, and the backbone structure of the prokaryotic vector was removed. Then the digested DNA was subjected to agarose gel electrophoresis, and the 18kb gel containing the target fragment to be recovered was cut out , Using a gel recovery kit (Omega) that can recover DNA fragments above 10kb, the results are as follows figure 2 Shown.

[0034] After dissolving the recovered fragments in TE solution (10mmol / L Tris.cl (pH7.4), 0.1mmol / LEDTA, prepared with Milli-Q water), measure the OD...

Embodiment 3

[0039] Example 3 Expression analysis of recombinant human BSSL in milk samples of transgenic mice

[0040] 1.1 Western blotting detection of transgenic mouse milk samples

[0041] The milk samples of F0 generation 9, 13, 14, 24, 25, and 29 mice were collected during lactation. Wild-type mouse milk was used as a negative control, and human milk was used as a positive control. The milk is collected on 8-12 days after the mouse is born. The mother mouse and the offspring are separated for more than 3 hours before collection, and a certain dose of oxytocin is injected into the abdominal cavity. The whey from which the milk fat and casein was removed after centrifugation was electrophoresed on a 10% SDS-PAGE and transferred to a nylon membrane. WesternBlot analysis with anti-human BSSL primary antibody can detect the expression of recombinant human BSSL in the milk of transgenic mice. Recombinant human BSSL has the same molecular weight as BSSL in human milk, about 100kDa, the results...

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Abstract

The invention provides a method for overexpression of cholate activation lipase in mammary gland of a mammal. The method comprises the following steps of transferring the gene coding human cholate activation lipase into the mammal so as to realize overexpression of human cholate activation lipase in the mammary gland of the mammal. According to the method, a transgenic mouse is prepared by utilizing construction of a BSSL (bile salt stimulation lipase) mammary gland specific expression vector; and human cholate activation lipase is overexpressed in the mammary gland of the mouse, the expression quantity of the human cholate activation lipase in milk of the transgenic mouse can achieve 0.38mg / ml, and the activity is 91473.94mU / ml. A premature young mouse is fed with transgenic milk of high expression cholate activation lipase, and the result shows that the survival rate of the premature young mouse is improved by 10% compared with that of the premature young mouse fed with milk of wild mouse. The method lays a foundation for producing human cholate activation lipase by utilizing a large livestock mammary gland bioreactor.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, in particular to a method for overexpression of bile salts in mammalian mammary glands to activate lipase. Background technique [0002] Bile salt-stimulated lipase (BSSL) is found to be present in the milk of humans, gorillas, cats, dogs, ferrets, and mice, but not in milk, and its activity can be found in the stomach of babies It remains stable under acidic conditions and pepsin hydrolysis conditions, and plays a very important role in the intestinal absorption of triglycerides in newborns. BSSL activity is stable under the pH environment of infant gastric juice. Bile salts protect it from inactivation of trypsin. BSSL cannot hydrolyze triglycerides in milk until it is mixed with bile salts in the duodenum. The reason why BSSL remains active under acidic gastric conditions and pepsin hydrolysis is mainly due to the oxygen-linked glycosylation repeat sites of BSSL. Because mucin plays a p...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/85C12N5/10C12N15/877A01K67/027
Inventor 王媛媛赵要风李宁
Owner BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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