A kind of truncated protein of Bombyx mori egg glue protein and application thereof

A technology of truncating proteins and colloids, which is applied in the fields of application, microbial-based methods, and the use of vectors to introduce foreign genetic materials, can solve the problems of research and application of undiscovered silkworm egg glue proteins, etc.

Active Publication Date: 2022-03-18
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant research and application of silkworm egg collagen as a bionic glue

Method used

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  • A kind of truncated protein of Bombyx mori egg glue protein and application thereof
  • A kind of truncated protein of Bombyx mori egg glue protein and application thereof
  • A kind of truncated protein of Bombyx mori egg glue protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, construct eukaryotic expression vector

[0023] 1.1 Target Fragment Synthesis

[0024] The eukaryotic expression fragment is based on the Bombyx mori villin BmEGP gene sequence, select the first 1500bp sequence (SEQ ID NO.1) of its sequence repeat region, add EcoR I at the 5' end, and add Not I at the 3' end for digestion Because the sequence is highly repetitive, common amplification is prone to base mutation and premature termination of amplification. Therefore, the entire sequence was chosen to be handed over to Qingke Bio for sequence synthesis, and the synthesized fragment was connected to the pUC57 vector for storage. The amino acid sequence corresponding to SEQ ID NO.1 is shown in SEQ ID NO.2.

[0025] 1.2 Target fragment EcoR I, Not I double digestion

[0026] The enzyme digestion system is as follows: Cut Smart 5 μL, EcoR I 1 μL, Not I 1 μL, pUC57-BmEGP plasmid 20 μL, ddH 2 O23 μL; enzyme digestion reaction conditions are: mix and centrifuge in...

Embodiment 2

[0041] Embodiment 2, Pichia pastoris competent preparation and transformation

[0042] 2.1 Yeast Competent Preparation

[0043] Recovery of X33 Competent Cells: Take 10 μL of preserved bacteria (X33), insert it into 10 mL of YPD liquid medium, use a 100 mL Erlenmeyer flask to culture overnight at 200 rpm in a constant temperature incubator at a temperature of 28 ° C to 30 ° C; use a pipette gun Dip the head with a small amount of overnight cultured bacteria solution, and in YPD (Kan + ) and grow at 28°C until the colonies are clearly visible (about 24-72h); pick a single colony, inoculate it in 5mL YPD liquid culture medium, and culture it in a 50mL Erlenmeyer flask at 250rpm overnight at 28°C to 30°C; It can be stored for several months, and the liquid can be stored for several weeks (stored at 4°C).

[0044] Take 100μL overnight X33 into 100mL YPD (Kan + ) liquid culture medium, in a 1L Erlenmeyer flask at 250rpm overnight at 28°C-30°C until the OD value is 1.3-1.5 (about...

Embodiment 3

[0049] Example 3, small amount of expression detection and large amount of eukaryotic recombinant protein

[0050] 3.1 Small expression detection

[0051] Put 25mL BMGY in a 250mL Erlenmeyer flask, inoculate the correct recombinant bacteria into it, place at 28°C, 250rpm, and cultivate until OD value = 2~6 (about 20~25h, OD=5 is the best) ); centrifuge at 1500g for 10min after the culture, discard the supernatant, add BMMY to the pellet to resuspend the yeast cells, and dilute to OD=1.0, transfer to a 1L Erlenmeyer flask for culture, and induce at 28°C and 250rpm Expression; every 24 hours, add 100% methanol to the medium in the ultra-clean workbench, so that the final concentration is 0.5%-1%, until 96 hours; take samples at regular intervals, and carry out sampling and cultivation every 0h, 24h, 48h, 72h, 96h Base 1mL, centrifuge at 8000-14000rpm at 4°C for 15min, collect the supernatant and precipitate separately, detect directly or freeze in liquid nitrogen and store at -...

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Abstract

The invention discloses an amino acid truncated protein of Bombyx mori villin. The nucleotide sequence of the villin is shown in SEQ ID NO.1, and the amino acid sequence of the villin is shown in SEQ ID NO.2. Link the sequence shown in SEQ ID NO.1 to a protein expression vector, and induce expression in yeast cells, separate and obtain crude ovalgelin, purify it through a nickel column, and then cross-link it with transglutaminase catalyzed to obtain The high-viscosity recombinant egg glue protein, through the shear tensile strength test, found that its viscosity reaches 2.2MPa, which is stronger than the viscosity of silkworm sericin (1.2MPa), even stronger than commercial glue PVP glue (1.2MPa), PVA glue (1.4 ~1.8MPa) and UHU glue (1.8MPa), the silkworm egg collagen produced by this method can be used to prepare biological glue with good adhesive strength.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a truncated protein of silkworm villin, a preparation method of the truncated protein of silkworm villin and an application of the truncated protein of silkworm villin as biological glue. Background technique [0002] Insects secrete sticky glue proteins that are used for various biological functions such as cocooning, anchoring eggs and capturing prey. Among them, the research on sericin in silk is particularly in-depth. Bombyx mori sericin is wrapped in the outer layer of silk fiber, which is used to bond the silk fiber into a cocoon shape, and make the cocoon attach to surrounding objects such as cocoons. Silkworm sericin is rich in serine, glycine, lysine, Threonine and other ingredients. Insects also secrete ovalolin in addition to sericin. Ovalgelin is synthesized and secreted by the mucous glands of adult females to anchor the eggs to the host plant. Li et al. (200...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/81C12N1/19A61L26/00A61L24/10C12R1/84
CPCC07K14/43586C12N15/815A61L24/108A61L26/0047C08L89/00
Inventor 董照明赵萍张艳雷雨田夏庆友
Owner SOUTHWEST UNIV
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