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59r mutant vector for expressing rfc protein and preparation method and application thereof

A protein and carrier technology, applied in the field of 59R mutant carrier and its preparation, can solve the problem of difficulty in obtaining soluble expression products, and achieve the effect of improving soluble expression ability and protein activity

Active Publication Date: 2021-11-30
BEIBU GULF UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the pSG system, rFC easily forms an insoluble complex with silk protein, and it is difficult to obtain soluble expression products

Method used

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  • 59r mutant vector for expressing rfc protein and preparation method and application thereof
  • 59r mutant vector for expressing rfc protein and preparation method and application thereof
  • 59r mutant vector for expressing rfc protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The present embodiment is the carrier construction of horseshoe crab C protein gene (rFC):

[0031] (1) Get the N-terminal sequence of the Nephila pilipes protein with better fiber performance on GeneBank, and use it as the wild-type sNT (gene sequence); carry out the 59th amino acid sequence of the wild-type sNT Mutated to obtain the sNTD59R sequence, the amino acid sequence of the mutated sequence is shown in the sequence table SEQ: NO: 1:

[0032] (2) Extract Limulus Chinese Limulus blood, clone Limulus Limulus C protein gene (rFC) and sequence, obtain Limulus Limulus C protein gene (rFC), its sequence is as shown in the sequence table SEQ: NO: 2:

[0033] Construct two vectors according to the above steps, and their ligation sequence is as follows: figure 1 and figure 2 shown:

[0034] in, figure 1 It is a schematic diagram of the construction of the wild-type vector (sNT+rFC): in the figure, the transposon of the piggyBac expression vector is connected with th...

Embodiment 2

[0037] This embodiment is the method for transferring the carrier of embodiment 1 into the silk gland:

[0038] (1) The two vectors of Example 1 are all injected into the embryo development zone of silkworm eggs by microinjection;

[0039] (2) After the silkworm eggs in step (1) are hatched and raised as worms, the moths with pure red-eye phenotype are screened for 3 consecutive generations, and the third-generation silkworms that do not segregate are selected for feeding, and the moths with pure red-eye gene are bred. transgenic silkworms that are homozygous or rFC protein homozygous and whose silk gland cells can secrete rFC protein;

[0040] (3) The rFC protein was secreted into the silk gland lumen of the silkworm under the action of the signal peptide of the heavy chain of silk fibroin, and then secreted into the cocoon.

[0041] In order to improve the hatching rate, you need to pay attention to the following points when injecting: ① Within the appropriate time range fo...

Embodiment 3

[0043] The rFC protein content is identified in the transgenic silk gland of embodiment 2, specifically as follows:

[0044] The silk gland of the 5th instar larvae of the transgenic silkworm was dissected, and the rFC protein was identified by Western Blot (WB); the results were as follows Figure 4 As shown in the figure, it can be seen that the binding fragment of rFC protein has appeared in the western blot, which proves that there is rFC protein in the silk gland. In the figure, the band of wild type (WT) is obviously thinner than that of 59R mutant type (MUT), This proves that the rFC protein expression level of the 59R mutant is much higher than that of the wild type in this experiment.

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Abstract

The invention relates to the field of transgenic technology, in particular to a carrier for expressing rFC protein and its preparation method and application; the carrier makes sNT insensitive to pH by mutating the sNT sequence, effectively solving the problem of exogenous Insolubility problems caused by protein fibrosis in the silk gland lumen; effectively prevent sNT monomers from forming dimers at low pH; improve the solubility of exogenous proteins such as rFC mediated by it in pSG bioreactors The expression ability and protein activity are a simple and efficient method for producing rFC protein, which effectively improves the protection of endangered wild animals.

Description

【Technical field】 [0001] The invention relates to the field of transgenic technology, in particular to a 59R mutation vector for expressing rFC protein, a preparation method and application thereof. 【Background technique】 [0002] Horseshoe crab is a very ancient marine organism, but its appearance has basically remained unchanged so far, so it is called the "living fossil" of the ocean. It occupies an important position in the biological chain of the marine intertidal zone and has extremely high ecological and economic value. . However, due to unrestrained fishing, at present, taking blood to extract the limulus reagent is one of the main reasons for unrestrained fishing of Limulus. The detection of endotoxin in biological products by Limulus reagent is the internationally recognized gold standard so far, and Limulus factor C (FC) in Limulus blood can replace Limulus reagent for the detection of endotoxin. Therefore, the efficient production of recombinant horseshoe crab ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/64C12N5/10C12N9/64
CPCC12N9/6408C12N15/64C12N15/8509C12N2015/8518C12N2800/103C12N2800/22C12Y304/21084
Inventor 王玉军黄精彩王红兰宇黄晓婵钟希发李依敏秦虹琳
Owner BEIBU GULF UNIV
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