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Method for increasing secretion rate of foreign protein expressed in silkworm cells

A technology of exogenous protein and secretion rate, applied in the field of genetic engineering, to achieve the effect of easy to understand, simple and clear method and low cost

Active Publication Date: 2021-02-09
上海五心大呈医疗科技(集团)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no literature reports or patent applications that apply the interference of target gene BiP to increase the secretion rate of exogenous protein expression in silkworm cells

Method used

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  • Method for increasing secretion rate of foreign protein expressed in silkworm cells
  • Method for increasing secretion rate of foreign protein expressed in silkworm cells
  • Method for increasing secretion rate of foreign protein expressed in silkworm cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1: The presence of GP64 SP affects the expression and secretion of foreign proteins

[0034]Transfect 2μg of recombinant plasmids pIZ / V5-SP-eGFP-TMD and pIZ / V5-eGFP-TMD into BmN cells respectively, add zeocin antibiotics 24 hours after transfection, and change the zeocin-containing medium every 3 days until fluorescence The ratio reaches 90%, and it can be stably expressed for a long time. Place the cell line under a fluorescence microscope (OLYMPUS IX 83) for observation, and compare the expression of eGFP in different cells with the same fluorescence intensity and exposure time. The result isfigure 1 As shown, compared with the control group pIZ / V5-eGFP-TMD, the presence of SP significantly reduced the expression of foreign protein eGFP (figure 1 : B). Using ImageJ-v1.8.0 to further measure its fluorescence intensity, it was found that the fluorescence intensity of SP was significantly lower than that of pIZ / V5-eGFP-TMD(figure 1 : C). The results show that in BmN cells,...

Embodiment 2

[0036]Example 2: Statistics of foreign protein expression, secretion and secretion rate after interference with host BiP

[0037]RNAi technology was used to knock down the expression of BiP in the host cells. The NC group was used as a control. Cell samples were collected after 24 hours, and the RNA was extracted. After reverse transcription, the expression of BiP was quantitatively analyzed by qPCR technology. The results showed that when siBiP was transfected, the relative expression of BiP in the host cells was only 22.48% of the control, and the expression was significantly reduced (figure 2: A).

[0038]After confirming that the expression of BiP in the host cell was effectively interfered, 0.8μg pIZ / V5-SP-Luc and pIZ / V5-Luc were transfected into BmN cells respectively, and the supernatant and luciferase in the cells were collected at different time points Activity, and calculate the secretion rate. The results showed that in the secreted protein (pIZ / V5-SP-Luc), when the expression ...

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Abstract

The invention relates to a method for increasing the secretion rate of foreign protein expressed in silkworm cells. An RNAi interference fragment for interfering a BiP gene in silkworm BmN cells is designed and synthesized, an expression vector carrying the foreign genes is transfected continuously after the BmN cells are transfected for 24 hours, and the secretion rate of the foreign protein canbe significantly increased after 72 hours. The method is simple and easy to operate, can effectively increase the secretion efficiency of the foreign protein expressed in the silkworm cells, can provide a new idea for improving the expression quantity of membrane protein by using BiP and the like as molecular targets, is beneficial to deep understanding of a baculovirus expression system, and provides a reference for mechanism research on participation of signal peptides in protein secretion.

Description

Technical field[0001]The invention belongs to the technical field of genetic engineering, and specifically relates to a method for improving the secretion rate of foreign protein expressed by silkworm cells after knocking down the target immunoglobulin heavy chain binding protein (BiP).Background technique[0002]With the improvement of human living standards and the enhancement of health awareness, the demand for recombinant protein drugs for various treatments or health care is increasing year by year, enabling the biotechnology industry to produce recombinant proteins for the treatment of vaccines, immunoglobulins, antibodies and protein factors. The growth rate of output value is strong, and its position in the biomedical industry is becoming more and more prominent.[0003]Many proteins need to be secreted outside the cell or located on the membrane to perform their biological functions. Many studies have shown that the expression efficiency of membrane proteins and secreted protei...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N15/85C12N15/67C12Q1/6888
CPCC12N15/87C12N15/85C12N15/67C12Q1/6888C12N2800/105C12Q2600/158
Inventor 郝碧芳刘娜孙璐萍徐瀛潘世佳沈兴家黄金山
Owner 上海五心大呈医疗科技(集团)有限公司
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