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Method for producing fluorescent antibacterial silks from transgenic silkworms

An antibacterial silk, transgenic technology, applied in the field of genetic engineering

Inactive Publication Date: 2012-12-26
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the method of obtaining silk protein fusion antimicrobial peptide and fluorescent protein (i.e., fluorescent antibacterial silk) using transgenic silkworms based on gene targeting

Method used

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  • Method for producing fluorescent antibacterial silks from transgenic silkworms
  • Method for producing fluorescent antibacterial silks from transgenic silkworms
  • Method for producing fluorescent antibacterial silks from transgenic silkworms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: A method for producing fluorescent antibacterial silk by transgenic silkworm (transgenic silkworm that expresses complete Fib-L and fuses GFP and silkworm Cecropin B)

[0054] The technical operation process is as follows:

[0055] 1. Cloning of the left arm of the gene targeting vector

[0056] The 1.2 kb left-arm primer sequence LL (SEQ ID No.1: ggt gag ctc gat caa act gca cac ggt gtg, underlined as Sac site) and LR (SEQ ID No.2: ggt tct aga gac gtg aac ctg gct ggc tg, underlined as Xba site), using the silkworm genome DNA as a template, the fragment obtained by PCR was cloned into the pMD19-T vector (for the method, refer to "Molecular Cloning", 1989, Science Press), and after sequencing verification, it was cloned into the pBluescriptⅡSK(+) vector (Stratagene company product) Sac / Xba site, and the obtained vector was named pSK-LL.

[0057] 2. Cloning of the right arm of the gene targeting vector

[0058] According to the publish...

Embodiment 2

[0077] Example 2: A method for producing fluorescent antibacterial silk by transgenic silkworm (transgenic silkworm that expresses part of the Fib-L gene and fuses GFP and silkworm Cecropin B)

[0078] The technical operation process is as follows:

[0079] 1. Cloning of the left arm of the gene targeting vector

[0080] The 1.2 kb left-arm primer sequence Fib-L-L1 (SEQ ID No.12: ttc gag ctc gtc gga cca gcc ctg ggt tg, underlined as Sac site) and Fib-L-L2 (SEQ ID No.13: ctg tct aga ttg acg atg cag tac tct tc, underlined as Xba The silkworm genomic DNA was used as a template, and the fragment obtained by PCR was cloned into the pMD19-T vector. After sequencing verification, it was cloned into the pBluescriptⅡSK(+) vector (product of Stratagene). Sac / Xba site, and the obtained vector was named pSK-FibLL.

[0081] 2. Cloning of the right arm of the gene targeting vector

[0082] The 0.5 kb right-arm primer sequence Fib-L-R1 (SEQ ID No.14) of the gene t...

Embodiment 3

[0096] Embodiment 3: A kind of transgenic silkworm that produces fluorescent antibacterial silk (expressing part of Fib-L gene, and the transgenic silkworm that fuses GFP and silkworm Cecropin B, DsRed for the negative selectable marker gene) method

[0097] The technical operation process is as follows:

[0098] 1. Cloning of the left arm of the gene targeting vector

[0099] Same as 1 in the implementation example two.

[0100] 2. Cloning of the right arm of the gene targeting vector

[0101] Same as implementation example 2 in 2.

[0102] 3. Construction of gene targeting vector

[0103] Same as 3 in the implementation example two.

[0104] 4. Construction of gene targeting vectors with negative selectable marker genes

[0105] In order to increase the frequency of gene targeting, a negative selection gene—red fluorescent reporter gene was introduced into the gene targeting vector DsRed . According to the published sequence (M76430) of GenBank, the specific pr...

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Abstract

The invention discloses a method for producing fluorescent antibacterial silks from transgenic silkworms. Gene targeting transgenic vectors fused with silkworm light-chain genes to express fluorescent proteins and antibacterial peptides are constructed on a vector of a homologous arm with silkworm silk fibroin light-chain genes; gene targeting is carried out through a transgenic method; the transgenic silkworms for producing fluorescent antibacterial silks are acquired after being subjected to the fluorescence screening and the identifying of molecular biology. According to the method disclosed by the invention, the quality of the silks produced from the silkworms is improved, the function of the silks is extended and the application value of the silks is increased; the fluorescent antibacterial silks are not polluted by DNA and other physical and chemical factors and have no potential harmfulness from transgenic organisms, so that the biological safety is high; and the simple preparation process and the low cost are achieved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for producing fluorescent antibacterial silk by using transgenic silkworms. Background technique [0002] For thousands of years, people have used the biological function of the silkworm to weave silk and form cocoons to produce a large amount of raw silk. Silk fiber is composed of protein. Although silk fibroin has many excellent performances, it also has some shortcomings. In order to improve the defects, in the transformation of silk, it mainly includes physical, chemical (post-processing) methods and genetic methods. Transformation method. In view of the characteristics of silk fiber, people hope to modify the silk gene at the source-gene level through genetic and molecular design, so as to improve the performance of silk fiber and expand the function of silk. Generally, the antibacterial effect of silk fiber mainly relies on the filtering effect of s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/63C07K19/00A01K67/04
Inventor 曹广力贡成良贾仲伟薛仁宇曾万仲郑小坚潘中华
Owner SUZHOU UNIV
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