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Modified human lactoferrin gene suitable for expression in silk gland of silkworm and its expression system and application

A human lactoferrin, gene technology, applied in the biological field, to control the risk of disease transmission and the effect of huge market development prospects

Active Publication Date: 2020-10-16
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2009, Wang Zhenxing disclosed the research on the expression of recombinant human lactoferrin using the silk gland bioreactor of silkworm, but this method can only express a small amount of protein in the middle silk gland of silkworm, and almost detect the expression of recombinant human lactoferrin in silkworm silk

Method used

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  • Modified human lactoferrin gene suitable for expression in silk gland of silkworm and its expression system and application
  • Modified human lactoferrin gene suitable for expression in silk gland of silkworm and its expression system and application
  • Modified human lactoferrin gene suitable for expression in silk gland of silkworm and its expression system and application

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Embodiment 1

[0030] Embodiment 1, transformation human lactoferrin gene synthesis

[0031] Download the amino acid sequence of the mature peptide of human lactoferrin (Human lactoferin, hLF, GenBank: M93150.1) from NCBI, and optimize the coding sequence according to the silkworm codon usage preference. As shown, the amino acid sequence is shown in the 1st to 700th positions of SEQ ID NO.2, and the gene sequence was synthesized by the company. Six histidine amino acids were fused to the C-terminus of the human lactoferrin (hLF) amino acid sequence to form hLF-his6. Both ends of the two synthetic recombinant protein gene sequences are respectively connected with BamHI and NotI restriction sites, as shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

Embodiment 2

[0032] Embodiment 2, the construction of transgene expression vector

[0033] The commercially synthesized hLF-his6 gene coding sequence was constructed into psl1180[hr3Pser1spRedSer1PA] through BamHI and NotI restriction sites to form psl1180[hr3CQSer1sphLFSer1], the nucleotide sequence is shown in SEQ ID NO.3, and then constructed through the AscI site into the AscI site of the pBac{3xp3EGFPaf} vector to form the transgene expression vector phShLFSer, the vector structure is as follows figure 1 shown.

Embodiment 3

[0034] Embodiment 3, microinjection and fluorescence screening

[0035] The transgene expression vector phShLFSer1 and the auxiliary carrier pHA3PIG plasmid were extracted using the QIAGEN Plasimd Mini Kit plasmid extraction kit, and the concentration of each plasmid was diluted to 400 ng / μl, and mixed with the auxiliary carrier pHA3PIG plasmid at a molar ratio of 1:1. Inject the mixed plasmid into Dazao early embryos that have been released from diapause (2-5 hours after oviposition), then seal the injection hole with non-toxic glue, sterilize with 35% formaldehyde steam for 5 minutes, and place at 25°C. Incubated in an environment with a relative humidity of 85%, the hatched larvae (G0 generation) were reared with artificial feed, and self-crossed or backcrossed to produce seeds after adulthood, and the obtained G1-generation silkworm eggs (7th day) were examined under a macroscopic stereofluorescence microscope. (Olypus MVX10, Japan), the red fluorescence observation adopts...

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Abstract

The invention relates to a modified human lactoferrin gene suitable for bombyx mori silk gland expression as well as an expression system adopting the modified human lactoferrin gene and applications.The modified human lactoferrin gene is as shown in SEQ ID NO.1, the amino acid coded by the gene is as shown in SEQ ID NO.2, the modified human lactoferrin gene, a secreting type sericin 1 gene promoter and a secreting type sericin 1 gene terminator form an expression cassette, an enhanser hr3 is adopted for enhancing the expression, meanwhile, a piggyBac transposition arm and a fluorescence selection marker gene are connected for constructing the expression system, and the expression system can efficiently express the recombinant human lactoferrin in bombyx mori silk gland, has biological activity, can be used for mass production of recombinant human lactoferrin, and has good market prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a modified human lactoferrin gene suitable for expression in silk glands of silkworms, and also relates to an expression system and application for expressing the human lactoferrin gene. 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 hotspot in the world t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/85C12N15/65
CPCC07K14/79C12N15/65C12N15/85C12N2830/001
Inventor 夏庆友王峰许胜王元成王日远陈文静赵萍
Owner SOUTHWEST UNIV
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