A method for large-scale production of high-purity porcine circovirus orf2 protein

A technology of porcine circovirus and ORF2, which is applied to the preparation method of peptides, biochemical equipment and methods, viruses, etc., can solve the problems of immune animal side effects and reduce vaccine efficacy, so as to solve side effects, improve processing efficiency, save cost effect

Active Publication Date: 2021-02-05
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These heterologous components not only cause side effects in immunized animals, but also greatly reduce the efficacy of vaccines

Method used

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  • A method for large-scale production of high-purity porcine circovirus orf2 protein
  • A method for large-scale production of high-purity porcine circovirus orf2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Antigen clarification pretreatment

[0048] Sterile chitosan was added to the porcine circovirus ORF2 protein culture solution in proportions of 1wt%, 5wt%, and 10wt%, respectively, after shaking well, it naturally settled at 4°C, and the supernatant was taken as the protein sample to be clarified.

[0049] Table 1 Evaluation of porcine circovirus ORF2 protein clarification pretreatment effect

[0050] sample Protein concentration (ug / ml) Miscellaneous protein removal rate Turbidity (FTU) Protein recovery Protein expression as is 11214 - 2000 - 1% Chitosan 5612 85.01％ 1000 99.8％ 5% Chitosan 4321 92.12％ 300 99.1％ 10% Chitosan 3123 93.50％ 50 91％

[0051] Use A 280 Methods The protein concentration was detected and the effective target protein content was detected by double antibody sandwich ELISA. The experimental results are shown in Table 1. The results in Table 1 show that the addition of chitosan can sign...

Embodiment 2

[0053] 2. Hollow fiber column clarification process

[0054] 1 system pretreatment

[0055] 1.1 Install the 0.2μm, 0.45μm, and 0.65μm hollow fiber columns into the hollow fiber column control equipment respectively, connect the corresponding pipelines, and soak the hollow fiber columns with sterile injection water for 10 minutes after assembly.

[0056] 1.2 System Integrity Detection

[0057] The pressure hold method checks the integrity of the system.

[0058] 1.3 System processing

[0059] Cleaning and sterilization: Use sterile 0.5mol / L NaOH solution to sterilize the system for 30 minutes, then clean the system with sterile water for injection to remove residual alkali solution until the pH is 7.0;

[0060] 1.4 Detection of water flux

[0061] Use sterile water for injection to pass through at the specified pump speed, and calculate the water flux of the hollow fiber column at the corresponding temperature.

[0062] 1.5 hollow fiber column balance

[0063] Equilibrate...

Embodiment 3

[0070] Embodiment 3 ion exchange column purification process

[0071] 1 system pretreatment

[0072] 1.1 Pound the ion-exchange filler Capto SP ImpRes into the BPG300 / 500 column, and measure the column efficiency after the ion-exchange column is assembled.

[0073] 1.2 Treat the molecular sieve gel chromatography column with sterile 0.5mol / L NaOH for 2 column volumes (CV), then wash with sterile injection water to pH 7.5, and then equilibrate the ion exchange column with 20mmol / L Tris solution to reach the conductivity The column is consistent before and after (the conductivity before and after the column is 6.5~6.6ms / cm), the pH is stable at 7.0, and the UV 280 The baseline is stable.

[0074] 2 purification process

[0075] Load the protein clarified solution, set the linear flow rate of the sample to 30-100cm / h, control the pressure to less than 2.50bar, and control the sample volume to 70-80mg / ml BSA (the maximum loading capacity of the filler is 95mg / ml BSA) . After ...

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Abstract

The invention specifically relates to a method for large-scale production of a high-purity porcine circovirus ORF2 protein, belonging to the technical field of vaccine preparation. The method comprises the following steps: weighing a porcine circovirus ORF2 protein culture solution, adding a small amount of chitosan and carrying out natural settling after shaking up; sucking up a supernatant and subjecting the supernatant to clarification and filtering with a hollow fiber column; then carrying out ion exchanging with an ion exchange column; and subjecting an eluate to desalination with a G25 chromatographic column, degerming and filtering successively so as to obtain high-purity porcine circovirus ORF2 protein liquid. According to the invention, large-scale (500 to 1000 L) high-efficiency production of the high-purity porcine circovirus ORF2 protein is realized through processes like settlement with chemical reagents, clarification and filtering, ion exchanging, and desalination with the G25 chromatographic column; the recovery rate of the high-purity porcine circovirus ORF2 protein can reach 99.46%; the removal rate of other proteins is as high as 98.04%; and the content of effective antigens is as high as 93.2%.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a method for large-scale production of high-purity porcine circovirus ORF2 protein. Background technique [0002] Porcine circovirus type 2 (PCV 2 ) contains two main open reading frames, wherein the virus capsid protein encoded by the ORF2 gene is the main structural protein, contains the main antigen neutralizing epitope, has good immunogenicity, and PCV 1 and PCV 2 There is no serological cross-reaction between them, so it is a good antigen for detecting the level of virus antibody and a good target gene for the development of new vaccines. The recombinant ORF2 protein (28kDa) expressed in vitro by the baculovirus expression system can self-assemble into viral nucleocapsid-like particles and exhibit good immunogenicity. Circovirus ORF2 protein has PCV 1 and PCV 2 Common immunogenicity is the current trend of vaccines. [0003] At present, conventional purific...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16
CPCC07K14/005C12N2750/10051
Inventor 周明光范金秀陈波徐高原金建云
Owner WUHAN KEQIAN BIOLOGY CO LTD
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