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A method for improving the secretion rate of exogenous protein expressed by silkworm cells

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

Active Publication Date: 2022-03-11
上海五心医疗科技集团股份有限公司
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  • Summary
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  • Description
  • Claims
  • Application Information

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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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  • A method for improving the secretion rate of exogenous protein expressed by silkworm cells
  • A method for improving the secretion rate of exogenous protein expressed by silkworm cells
  • A method for improving the secretion rate of exogenous protein expressed by 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 BmN cells with 2 μg of recombinant plasmids pIZ / V5-SP-eGFP-TMD and pIZ / V5-eGFP-TMD respectively, add zeocin antibiotics 24 hours after transfection, and replace the medium containing zeocin every 3 days until fluorescence The ratio reaches 90%, and it can be expressed stably for a long time. The cell lines were observed under a fluorescence microscope (OLYMPUS IX 83), and the expression of eGFP in different cells was compared using the same fluorescence intensity and exposure time. The result is as figure 1 As shown, compared with the control group pIZ / V5-eGFP-TMD, the presence of SP significantly reduced the expression of exogenous 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 resul...

Embodiment 2

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

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

[0038] After confirming that the expression of BiP in the host cells was effectively interfered, 0.8 μg 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 secretory protein (pIZ / V5-SP...

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Abstract

A method for improving the secretion rate of exogenous protein expressed by silkworm cells, designing and synthesizing an RNAi interference fragment for interfering with the BiP gene in silkworm BmN cells, transfecting BmN cells for 24 hours and then continuing to transfect the expression vector carrying the foreign gene, after 72 hours That is, the secretion rate of the foreign protein can be significantly increased. This method is simple and easy to operate, and can effectively improve the secretion efficiency of exogenous proteins expressed in silkworm cells. It can provide a new idea to improve the expression of membrane proteins by using BiP etc. as molecular targets, and is conducive to in-depth understanding of baculovirus Expression system, and provide a reference for the mechanism research of signal peptide involved in protein secretion.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for increasing the secretion rate of exogenous protein expressed by silkworm cells after knocking down 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 various therapeutic or health-care recombinant protein drugs is increasing year by year, making the biotechnology industry that produces therapeutic recombinant proteins such as 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 to the outside of the cell or located on the membrane to exert their biological functions. Many studies have shown that the expression efficiency of membrane prote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 上海五心医疗科技集团股份有限公司
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