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Vero cell cracked protein, preparation method and application thereof

A technology for lysing proteins and cells, applied in the field of preparation, Vero cell lysing proteins, which can solve the problems of substances that are difficult to prepare and not sold, and achieve the effects of good repeatability, strong specificity, and high sensitivity

Active Publication Date: 2009-11-04
YUXI WALVAX BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no kit for detecting HCP residues in Vero cells on the market. The main reason is that several substances required in the detection process of HCP residues are difficult to prepare: one of them is Vero cell lysate protein, which can be used as an immune Antibodies produced by the original immunized animals - the antibodies can be used for subsequent detection reactions; and play the role of test standards in the detection process

Method used

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  • Vero cell cracked protein, preparation method and application thereof
  • Vero cell cracked protein, preparation method and application thereof
  • Vero cell cracked protein, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: the preparation of Vero cell lysate protein

[0036] Source of material:

[0037] Vero cell working seed cell: ATCC serial number is No: CCL-81

[0038] Main equipment:

[0039] Cell ultrasonic breaker: SONICS VCF1500, the maximum output power is 1500W

[0040] Ultrafilter: Pellicon 2Cassette Filter; (Millipore Company);

[0041] Ultrafiltration membrane, Biomax-100A (MWCO=100KD) (Millipore Company);

[0042] Ultrafiltration concentrator: (MWCO=100KD) (Millipore Company);

[0043] Chromatography purifier: (GE Healthcare)

[0044] Column: BPG 140 / 950; INdEX 140 / 500 (GE Healthcare)

[0045] Purification medium: Phenyl Sepharose 6Fast Flow hydrophobic gel (GE Healthcare)

[0046] Sepharose 4Fast Flow molecular sieve gel (GE Healthcare company)

[0047] Preparation method: see figure 1

[0048] 1. Harvesting of cell culture: Take a cell tube of Vero cell line in the working cell bank, inoculate the cells into a cell culture flask, add 199 medium co...

Embodiment 2

[0059] Example 2 Preparation of polyantibody against Vero cell lysate protein

[0060] Source of material:

[0061] Purified Vero cell lysate protein was derived from Example 1.

[0062] experimental animals

[0063] Rabbit (common grade) comes from Shanghai Puxin Biological Company

[0064] Guinea pigs (clean grade) are from Shanghai Experimental Animal Center, Chinese Academy of Sciences

[0065] Other instrument reagents

[0066] Protein-A affinity chromatography column, goat anti-rabbit-HRP and goat anti-guinea pig-HR were purchased from KPL Company; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma Company.

[0067] 1. Immunization of rabbits:

[0068] Three rabbits were immunized, each rabbit was first immunized with 0.5 mg Vero cell lysate protein prepared in Example 1, added with an equal volume of Freund's complete adjuvant, after emulsification was sufficient, the back was subcutaneously injected at 6-8 points. The second imm...

Embodiment 3

[0087] Embodiment 3: The establishment of ELISA double antibody sandwich method:

[0088] Coating: Dilute the guinea pig anti-Vero cell lysing protein IgG prepared in Example 2 to 5-20 μg / ml with 0.05 mol / LCB (pH 9.6), coat the microplate, 100 μl / well, overnight at 4°C. The next day, wash twice with 0.01mol / L PBS (pH 7.4), add 150 μl of 2% BSA-PBS to each well, and block at room temperature for 2 hours. Wash twice with PBS, air-dry, and seal with a ziplock bag with a small packet of desiccant inside each bag. Store at 4°C for later use.

[0089]Add sample: Dilute the Vero cell lysate protein standard prepared in Example 1 with PBS-T to 4000ng / ml, 2000ng / ml, 1000ng / ml, 500ng / ml, 250ng / ml, 125ng / ml, 62.5ng / ml. Add 100 μl of the above-mentioned standard, 0.01mol / L PBS-T and the sample to be tested to each well. React at 37°C for 60 min, wash with PBS-T 4 times, and pat dry.

[0090] Add HRP-rabbit anti-Vero cell lysis protein IgG: Dilute the HRP-rabbit anti-Vero cell lysis pr...

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Abstract

The invention belongs to biotechnology field, in particular relates to a Vero cell cracked protein, a preparation method and an application thereof. The invention discloses a method of preparing the Vero cell cracked protein, and the method comprises the following steps: culturing and harvesting Vero cells; cracking the Vero cells; purifying the Vero cell cracked protein; and concentrating to obtain Vero cell cracked protein. The invention also discloses the Vero cell cracked protein prepared by the method and the application thereof. The Vero cell cracked protein can be used as immunogen to establish a Vero cell HCP test method and as a standard product of tests, has wider range of application, high sensitivity and good repetitiveness and can be applied to quality control and analysis inthe preparation process of Vero cell produced vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to Vero cell lysate protein, a preparation method and application thereof. Background technique [0002] Vero cells (African green monkey kidney cells) are an ideal matrix for vaccine production, with clear genetic background, stable karyotype, no exogenous factor contamination, suitable for large-scale cultivation, and can be produced in bioreactors, ensuring mass production of vaccines homogeneity and safety. Over the years, a variety of vaccines produced by Vero cells have been successfully developed abroad and approved for marketing, including purified human rabies vaccine, inactivated (purified) polio vaccine (IPV), inactivated Japanese encephalitis vaccine (JE ). There are two production methods for vaccines produced by Vero cells: one is the secretory virus culture method (after virus replication is completed, it is secreted into the extracellular culture medium); i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C07K1/34G01N33/68
Inventor 史晋施松明姚越刘毅
Owner YUXI WALVAX BIOTECH CO LTD