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Gene for modifying human acidic fibroblast growth factor applied to silkworm silk gland expression, and expression system and application of gene

A fibroblast and growth factor technology, applied in the field of genetic engineering, can solve the problems of restricting large-scale promotion and application, and demanding operating cost environment

Active Publication Date: 2014-07-09
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using Chinese hamster ovary cells as a bioreactor to produce foreign proteins is currently the most standard expression mode, but its operating costs and environmental requirements are extremely harsh, which severely limits its large-scale application

Method used

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  • Gene for modifying human acidic fibroblast growth factor applied to silkworm silk gland expression, and expression system and application of gene
  • Gene for modifying human acidic fibroblast growth factor applied to silkworm silk gland expression, and expression system and application of gene
  • Gene for modifying human acidic fibroblast growth factor applied to silkworm silk gland expression, and expression system and application of gene

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Experimental program
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Effect test

Embodiment 1

[0034] 1. Gene synthesis

[0035] The amino acid sequence of human acidic fibroblast growth factor (haFGF) was downloaded from NCBI, and the coding sequence was optimized and designed according to the codon usage preference of Bombyx mori. The C-terminus of the sequence is fused with 6 histidine amino acids to form haFGF-his 6 , and in haFGF-his 6 The two ends of the nucleotide sequence are respectively connected with BamHI and NotI restriction sites, that is, the sequence shown in SEQ ID NO.1, and the amino acid sequence encoded by the expression frame is shown in SEQ ID NO.2.

[0036] 2. Construction of transgene expression vector

[0037] Artificially synthesized haFGF-his designed by codon optimization 6 The double strand of the coding sequence (SEQ ID NO.1) was digested with BamHI and NotI to replace the red fluorescent protein gene Red in the psl1180[hr3Pser1spRedSer1PA] vector (disclosed in Chinese Patent Publication No. 102492692A) to obtain psl1180[hr3CQSer1sphaFGF...

Embodiment 2

[0040] Example 2 Tissue localization of exogenous protein in sghaFGF-his6 silk gland in silkworm middle part and silk

[0041] The middle silk glands and silks of sghaFGF silkworm and normal silkworm on the 6th day of fifth instar were fixed in 4% paraformaldehyde for 30 minutes, then rinsed with 1×PBS for 10 minutes three times, and then washed with 70% ethanol for more After that, they were stored at 4°C in 70% ethanol by volume. Subsequently, the silk gland tissue was embedded in paraffin, and the embedded tissue material wax block was sliced ​​with a Leica RM2235 paraffin microtome with a slice thickness of 4-7 μm. Finally, the wax tape was segmented and glued to the anti-off glass slide coated with poly-lysine. use O.C.T. TM The reagents were used to embed silk samples, and the samples were cross-cut into 10 μm thin slices in a cryostat. The flakes were collected on glass slides for immunoblotting, the primary antibody was anti-FGF1 polyclonal antibody, the secondary...

Embodiment 3

[0043] 1. Detection of recombinant human acidic fibroblast growth factor (haFGF) content in total cocoon shell protein

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Abstract

The invention discloses a gene for modifying a human acidic fibroblast growth factor applied to silkworm silk gland expression, and an expression system and application of the gene. The gene for modifying the human acidic fibroblast growth factor is represented by the 7-450 site of SEQ ID NO.1; the amino acid coded by the gene is represented by SEQ ID NO.2; the gene for modifying the human acidic fibroblast growth factor, a gene promoter of secretory type sericin 1 and a gene terminator of the secretory type sericin 1 form an expression box, and the expression box is expressed by using an enhancer hr3 in an enhanced manner; simultaneously, the expression system is constructed by accessing a piggyBac transposable arm and a fluorescent selected and marked gene. The expression system is capable of expressing and recombining the human acidic fibroblast growth factor in silkworm silk gland at high efficiency; furthermore, the expression system has biological activity and can be used for producing and recombining the human acidic fibroblast growth factor in a large scale, therefore, the expression system has good market prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a modified human acidic fibroblast growth factor gene suitable for expression in silk glands of silkworms, and also relates to an expression system containing the gene and its application. Background technique [0002] Since the beginning of the 21st century, with the increasing demand for functional proteins for various purposes such as medical, medicinal, edible, beauty, and health care, relying on extraction and production of proteins from natural sources has been unable to meet the rapidly growing market demand. . Establish and improve various high-efficiency prokaryotic and eukaryotic expression systems, and use E. coli, yeast, insect cells, mammalian cells, insects and mammals as host bioreactors to achieve low-cost, large-scale production with biological activity An effective and sustainable method for recombining foreign proteins, and has become a research ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85C07K14/50C07K1/22
Inventor 夏庆友王峰徐汉福王日远赵萍
Owner SOUTHWEST UNIVERSITY
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