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Method for constructing anti-mammitis transgenic mouse model and special vector for method

A mastitis and gene technology, applied in the field of construction method of anti-mastitis transgenic mouse model and its special carrier, can solve the problems of bacterial drug resistance and antibiotic residue

Inactive Publication Date: 2014-07-16
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the treatment of mastitis at home and abroad is still dominated by antibiotics, but long-term use of antibiotics will lead to many unfavorable factors such as bacterial resistance and antibiotic residues

Method used

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  • Method for constructing anti-mammitis transgenic mouse model and special vector for method
  • Method for constructing anti-mammitis transgenic mouse model and special vector for method
  • Method for constructing anti-mammitis transgenic mouse model and special vector for method

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, transgenic recombination vector construction

[0040] The transgenic recombination vector pBC1+adapter+seq2+IRES-seq3 is a vector obtained by inserting sequence 1 in the sequence table between Sac1 and Apa1 of pBC1+adapter, and it can also be named plasmid pBC1-Lysotaphin-peptidoglycan endolysin B30.

[0041] Among them, sequence 1 consists of 2065 nucleotides, lysostaphin from the 1st to 799th nucleotides at the 5' end, and lysostaphin from the 800th to 1469th nucleotides at the 5' end is internal ribosome entry The site (Internal ribosome entry site, IRES), the 1470-2065 nucleotides from the 5' end is peptidoglycan endolysin (Peptidoglycan endolysin B30).

[0042] The pBCl vector uses the goat β-casnein promoter to guide the high-efficiency and specific expression of the recombinant protein in mammary gland tissue, and was purchased from Invitrogen Company, Cat. No. K27001.

[0043] Internal ribosome entry site (Internal ribosome entry site, IRES), IRES c...

Embodiment 2

[0058] Embodiment 2, construction of anti-mastitis transgenic mouse model

[0059] 1. Obtaining DNA Fragments by Microinjection

[0060] Take about 2 μL of the plasmid pBC1+adapter+seq2+IRES-seq3 obtained in Example 1, add restriction enzymes AatII and NarI, restriction enzyme reaction buffer, add water to make the volume to 40 μL, flick the tube wall Mix evenly, place in a 37°C incubator for 1 hour, and then take 3 μL of the reaction solution for 1% agarose gel electrophoresis to observe the enzyme digestion results.

[0061] The reaction system for enzyme digestion of transgene vectors is shown in Table 1:

[0062] Table 1 Reaction system for enzyme digestion of transgenic vectors

[0063]

[0064] The results of DNA agarose gel electrophoresis figure 1 As shown, 1: AatII and NarI digested fragments; M: 1000bp DNAladder, recovering 18.5kb digested fragments to obtain microinjected DNA fragments, after sequencing, the microinjected DNA fragments include sequence 1 in th...

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Abstract

The invention discloses a method for constructing an anti-mammitis transgenic mouse model and a special vector for the method. An anti-mammitis-related protein composition comprises lysostaphin and peptidoglycan endolysin B30; the peptidoglycan endolysin B30 derives from a Streptococcus agalactiae phage B30, and an amino acid sequence of the peptidoglycan endolysin B30 is sequence 2 in a sequence table; and an amino acid sequence of the lysostaphin is sequence 3 in the sequence table. The experiment proves that a transgenic plasmid for mammary gland-specific expression of the lysostaphin and the peptidoglycan endolysin B30 is constructed by using a mammary gland-specific expression vector; and the anti-mammitis transgenic mouse model is constructed by a microinjection method, a transgenic mouse with an anti-bovine mastitis gene is obtained, and a technical model is provided for further producing anti-mammitis transgenic calves.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing an anti-mastitis transgenic mouse model and a special carrier thereof. Background technique [0002] Cow mastitis is a complex and serious common disease of dairy cows. Its incidence rate is very high, and the difficulty of control is also increasing. Despite years of research worldwide, it remains by far one of the most costly diseases in dairy farming, severely hindering the development of the dairy industry. The economic losses caused by mastitis are due to the fact that the milk production capacity of affected cows is much lower than that of normal cows; the milk quality of affected cows is reduced due to changes in milk composition, resulting in discarded milk; the pregnancy time and estrus cycle of affected cows are prolonged; the elimination of cows The increase in the rate and the cost of the treatment of the sick cows, etc., and the decline in milk...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01K67/027A61K49/00
Inventor 张建方俊顺苗丽云罗玲娟
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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