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Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo

A plasmid carrier and construction method technology, applied in recombinant DNA technology, using vectors to introduce foreign genetic material, animal husbandry, etc., can solve problems such as environmental pollution, high energy consumption of chemical methods, and inability to change the biochemical characteristics of silk fibroin, etc., to achieve The effect of reducing energy consumption and reducing steps

Inactive Publication Date: 2014-12-17
JIANGSU UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to breed a new type of transgenic silkworm, which has a recombined silk fibroin heavy chain protein gene, and this recombined silk fibroin heavy chain protein contains an MMP enzyme cleavage site, which solves the problem of energy consumption in chemical methods. Many, environmental pollution problems, physical methods can not change the biochemical properties of silk fibroin

Method used

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  • Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo
  • Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo
  • Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo

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Embodiment 1

[0021] The invention adopts gene cloning technology to construct a novel silkworm transgenic plasmid carrier, the plasmid carrier has a recombined silk fibroin heavy chain protein gene, and the recombined silk fibroin heavy chain protein contains an MMP restriction site. Specific steps are as follows:

[0022] 1. Design a new silkworm transgenic plasmid vector construction process

[0023] Design is used to construct the process flow of a kind of novel silkworm transgenic plasmid vector, see figure 1 . Among them, fibroin H refers to silk fibroin heavy chain gene, PCR refers to polymerase chain reaction technology, FibH-N refers to silk fibroin heavy chain protein N-terminal gene sequence, A, B, F refer to Asc I, BamH I, Fse I enzyme digestion site, pMD-FibH-N refers to the pMD-18T simple vector with FibH-N cloned (purchased from Takara Company), and FibH-GS-site-C refers to the C-terminus of silk fibroin heavy chain protein with MMPs restriction site gene sequence.

[002...

Embodiment 2

[0073] In the present invention, the constructed novel silkworm transgenic plasmid vector pBac[3×P3-EGFPafm]-FibH-GS-site is mixed with Piggybac Help Plasmid in equal amounts, and the mixed plasmid is injected into silkworms that have been born for a certain period of time by microinjection. in the egg. Since the 3×P3 promoter is used in the transgenic vector, the success of the transgene is identified by observing the fluorescence phenomenon in the eyes of the silkworm moth, and the obtained transgenic silkworm is identified by genome PCR and sequencing. Specific steps are as follows:

[0074] 1 plasmid extraction

[0075] The plasmid was extracted using the kit QIAprep Spin Miniprep Kit (Product No.: 27104), and the operation was strictly in accordance with the instructions of the plasmid extraction kit. (Note: Add LyseBlue reagent to Buffer P1. Add the RNase A solution included in the kit to Buffer P1, mix, and store P1 at 2-8°C. When using Buffer PE, add absolute ethanol...

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Abstract

The invention relates to a novel genetically modified plasmid vector, its construction and a cultivation method of silkworms containing a novel recombination gene in vivo. The novel genetically modified silkworm has a recombinant fibroin heavy chain protein gene containing MMPs enzyme sites. The invention belongs to the field of agricultural insect excellent variety cultivation and utilization. A flow diagram for construction of the novel genetically modified vector is as shown in the attached map. By the characteristic that the MMPs enzyme sites can undergo MMPs enzyme digestion, the MMPs enzyme sites are introduced into silkworm fibroin. Thus, the silkworm fibroin has new properties and the purpose of modifying silkworm fibroin through a biological method is achieved. According to the patent, the recombinant fibroin protein gene is cloned to a PiggyBac transposon vector; the vector is injected into silkworm eggs through a micro-injection method; after incubation of the silkworm eggs, individuals with green fluorescence eyes are selected so as to determine the successfully-cultivated novel genetically modified silkworms.

Description

technical field [0001] The invention belongs to the field of cultivating and utilizing fine varieties of agricultural insects, and in particular relates to the field of a method for cultivating novel recombinant gene silkworms. Background technique [0002] As a natural material, silk has strong advantages in the field of biomedicine, such as biocompatibility, safety, degradability, good mechanical properties, etc. As a medical material, silk has many requirements and also has some limitations. For example, as a tissue engineering scaffold, its degradation rate is required to be consistent with the growth rate of tissue cells, but the existing silk cannot be better controlled in a suitable time. degraded; as a drug delivery system, the identification of the drug action site on silk itself is very low, if silk has a better guiding effect, it will play a greater role in the drug delivery process; when silk participates in the treatment of tumors and other diseases , as a sens...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66A01K67/04
Inventor 黄国平孙春凤陈克平姚勤陈慧卿张春霞张志燕
Owner JIANGSU UNIV
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