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Method for detecting residual quantity of Pichia pastoris host protein in recombinant human lysozyme

A Pichia pastoris and host protein technology, applied in the biological field, can solve the problem of not finding Pichia pastoris host protein residues, etc., and achieve the effects of no cross-reaction, simple method, and complete antibody coverage.

Active Publication Date: 2019-05-24
SHAANXI HUIKANG BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is clearly stipulated in the Pharmacopoeia that the method for the determination of yeast cell protein residues is the enzyme-linked immunoassay method, but after searching the market, no commercial enzyme-linked immunosorbent assay kits have been found for the detection of Pichia host protein residues

Method used

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  • Method for detecting residual quantity of Pichia pastoris host protein in recombinant human lysozyme
  • Method for detecting residual quantity of Pichia pastoris host protein in recombinant human lysozyme
  • Method for detecting residual quantity of Pichia pastoris host protein in recombinant human lysozyme

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Effect test

Embodiment 1

[0051] The steps of the detection method for the residual amount of Pichia host protein in the recombinant human lysozyme raw material of the present embodiment are as follows:

[0052] (1) Preparation of recombinant human lysozyme

[0053] Use an electric shock transformation instrument to transform 10ng of pPIC9K-hLYZ recombinant plasmid into 100uL Pichia pastoris GS115 competent by electric shock according to the operation method of the instrument, use the histidine deficiency screening method and resistance screening method to obtain the expression strain, induce fermentation and purify Recombinant human lysozyme.

[0054] (2) Preparation of Pichia pastoris host protein

[0055] Use an electric shock transformation instrument to transform 10ng of pPIC9K empty plasmid into 100uL Pichia pastoris GS115 competent by electric shock according to the operation method of the instrument, use the histidine deficiency screening method and resistance screening method to obtain positi...

Embodiment 2

[0087] The steps of the detection method for the residual amount of Pichia host protein in the recombinant human lysozyme raw material of the present embodiment are as follows:

[0088] Steps (1) to (4) are the same as in Example 1.

[0089] (4) Eliminate cross-reaction between recombinant human lysozyme and Pichia host protein immunized rabbit serum and Pichia host protein immunized sheep serum

[0090] 1) Preparation of polyclonal antibodies

[0091] Take 1mL of rabbit serum, add 2ml of 0.06mol·L -1 pH4.8 sodium acetate buffer, then add 20ul n-octanoic acid to mix, rabbit serum and 0.06mol·L -1 Sodium acetate buffer solution with pH 4.8, n-octanoic acid in a volume ratio of 1:2:0.02, centrifuged at 10,000 rpm, and the supernatant was taken; use 1mol L -1 Adjust the pH to 7.4 with NaOH solution, add ammonium sulfate solution with a mass concentration of 40% until the protein precipitates, centrifuge at 10000rpm for 20min to collect the precipitate, add 1ml of 0.1mol·L -1 ...

Embodiment 3

[0101] The steps of the detection method for the residual amount of Pichia host protein in the recombinant human lysozyme raw material of the present embodiment are as follows:

[0102] Steps (1) to (4) are the same as in Example 1.

[0103] (5) Eliminate the cross-reaction between recombinant human lysozyme and Pichia host protein immune rabbit serum and Pichia host protein immune sheep serum

[0104] 1) Preparation of polyclonal antibodies

[0105] Take 1mL of rabbit serum, add 2ml of 0.06mol·L -1 pH4.8 sodium acetate buffer, then add 50ul n-octanoic acid to mix, rabbit serum and 0.06mol·L -1 Sodium acetate buffer solution with pH 4.8, n-octanoic acid in a volume ratio of 1:2:0.05, centrifuged at 10,000 rpm, and the supernatant was taken; use 1mol L -1 Adjust the pH to 7.4 with NaOH solution, add ammonium sulfate solution with a mass concentration of 40% until the protein precipitates, centrifuge at 10000rpm for 20min to collect the precipitate, add 1ml of 0.1mol·L -1 Ph...

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Abstract

The invention discloses a method for detecting residual quantity of Pichia pastoris host protein in a recombinant human lysozyme raw material. The method includes the steps of preparing recombinant human lysozyme; preparing Pichia pastoris host protein; detecting total protein content of the Pichia pastoris host protein; preparing antiserum of the Pichia pastoris host protein; eliminating a crossreaction between the recombinant human lysozyme and immune rabbit serum of the Pichia pastoris host protein and immune sheep serum of the Pichia pastoris host protein; preforming two-dimensional electrophoresis on the Pichia pastoris host protein; detecting antibody coverage rate of the Pichia pastoris host protein; and preforming enzyme-linked immunosorbent assay. By adopting the detection method, the Pichia pastoris host protein in the recombinant human lysozyme is continuously detected for 3 batches, and the content of the Pichia pastoris host protein is all less than 0.05%, and meets the standard of less than 0.1% specified in the Pharmacopoeia of the People's Republic of China. The detection method has the advantages of strong specificity, complete antibody coverage rate, no cross reaction and the like, and can be used for detecting the content of the Pichia pastoris host protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection method for detecting the residual amount of Pichia pastoris host protein in recombinant human lysozyme raw materials. Background technique [0002] Dry eye syndrome refers to the general term for various diseases that are caused by abnormal tear quality or quantity or dynamics caused by any reason, resulting in decreased tear film stability, accompanied by eye discomfort or ocular surface tissue lesions. With the popularization of video terminal products, the proportion of young and middle-aged people suffering from dry eye syndrome has increased significantly. In recent years, dry eye patients in cities are rapidly increasing at a rate of 10% to 20% per year. [0003] The Pichia pastoris expression system has been developed for 40 years since it was developed in the 1980s. It is a complete set of exogenous gene expression system, which is easy to ferment at ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/561
Inventor 李晓颖高恩史瑾侯增淼陈沛王鑫杨小琳赵金礼
Owner SHAANXI HUIKANG BIO TECH CO LTD
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