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59R mutant vector for expressing rFC protein as well as preparation method and application of 59R mutant vector

A protein and vector technology, applied in the field of 59R mutant vector and its preparation, can solve the problems such as difficulty in obtaining soluble expression products

Active Publication Date: 2021-02-09
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 as well as preparation method and application of 59R mutant vector
  • 59R mutant vector for expressing rFC protein as well as preparation method and application of 59R mutant vector
  • 59R mutant vector for expressing rFC protein as well as preparation method and application of 59R mutant vector

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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 properties 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 with 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 technical field of transgenosis, in particular to a vector for expressing rFC protein as well as a preparation method and application of the vector. According to the vector, an sNT sequence is mutated, so that sNT is insensitive to pH, and the problem of insolubility caused by fibrosis of foreign protein in a silk gland cavity in a pSG expression system is effectivelysolved. The condition that an sNT monomer forms a dimer at a low pH value, can be effectively avoided. The soluble expression capability and the protein activity of rFC and other foreign proteins mediated by the vector in a pSG bioreactor are improved. The method is a simple and efficient rFC protein production method, and the protection on endangered wild animals is effectively improved.

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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IPC IPC(8): C12N15/85C12N15/64C12N5/10C12N9/64
CPCC12N9/6408C12N15/64C12N15/8509C12N2015/8518C12N2800/103C12N2800/22C12Y304/21084
Inventor 黄精彩王红兰宇黄晓蝉钟希发李依敏秦虹琳王玉军
Owner BEIBU GULF UNIV
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