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Method for taking off endotoxin in primary pure plasmids or proteins, and kit

A protein and endotoxin technology, applied in the field of biomedicine, can solve the problems of unreachable, affected yield, and poor removal of plasmid endotoxin.

Inactive Publication Date: 2007-12-26
SHANGHAI H & G BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, this gel chromatography or mixing method will inevitably dilute the plasmid solution. Even if the removal of endotoxin meets the requirements, at least one concentration step must be added, which will also affect the yield.
At present, Beijing Zhuoguan Technology Co., Ltd. has produced agarose gel FF (covalently coupled polymyxin agarose gel) for endotoxin removal, but because its adsorption endotoxin amount is lower than 10,000 / ml, crude The content of endotoxin in the extracted plasmid is very high, so the effect of removing plasmid endotoxin is not good, and it cannot meet the requirement of less than 100EU / mg
[0015] All mention PMXB gel chromatography in the literature about adopting PMXB to remove endotoxin, there is no use of PMXB to carry out organic solvent centrifugal phase separation to remove endotoxin report or kit
Moreover, there is no report that the complex formed by PMXB combined with endotoxin is more soluble in organic solvents

Method used

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  • Method for taking off endotoxin in primary pure plasmids or proteins, and kit

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Experimental program
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specific Embodiment approach

[0041] The content of the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments, and the present invention will be further elaborated, but these embodiments are by no means limiting the present invention. Any changes made by those skilled in the art in the implementation of the present invention under the inspiration of this specification will fall within the scope of the claims of the present invention.

[0042] Experimental Materials and Instruments

[0043] Primary purified plasmids: HG43, PSW, PSA43 and Ag85A recombinant protein solutions were obtained from Shanghai Haigui Biotechnology Co., Ltd.

[0044] Organic solvents: isopropyl myristate, ethyl acetate, diethylene glycol monomethyl ether, benzyl benzoate, chloroform, Analytical grades were purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company. Polymyxin B sulfate (PMXB) and Triton-X-114 were purchased from Sigma (P1004 a...

Embodiment 1

[0048] Example 1 Determination of endotoxin content in plasmid liquid by semi-quantitative method for gelation of limulus reagent

[0049] 1. Recheck the sensitivity of LAL reagent : Adopt the Limulus test kit (sensitivity is 0.25EU / ml) and endotoxin standard substance (10EU / ml) produced by Zhanjiang Andus Company, operate according to the manufacturer's instructions, and the check results are shown in Table 1.

[0050] Table 1 Limulus Reagent Sensitivity Review

[0051] endotoxin

Standard solution concentration

4λ

1EU / ml

2λ

0.5EU / ml

lambda

0.25EU / ml

0.5λ

0.125EU / ml

Negative

sex

gelled result

++++

++++

++++

----

--

[0052] The above experimental results show that the sensitivity of the Limulus reagent used is consistent with the marked value, and it is used according to the marked value.

[0053] 2. Serial dilution of plasmid sample...

Embodiment 2

[0061] Example 2 Triton Fog Point Centrifugal Phase Separation Method for Removing Endotoxin in Plasmid Liquid

[0062] This example is to repeat the steps described in the literature (Cotten, Baker A, Sallik M, et al.: GeneTherapy, 1: 239-46, 1994), in order to verify the effect of this method on removing endotoxin.

[0063] Take 1ml of the initial pure HG43 plasmid solution with a concentration of 1.2mg / ml (prepared with 0.1M PBS buffer solution) and add it to a clean centrifuge tube. Add TritonX-114 (undiluted). Mix at 4-10°C for 15 minutes, then move to 35-37°C and let it stand for 5 minutes. After the mixture becomes cloudy and turbid, centrifuge at 13,000rpm in a desktop centrifuge at the same temperature for 5 minutes, and oily liquid deposits at the bottom of the small tube (for TritonX -114 complex with endotoxin). Carefully draw the upper aqueous phase containing the plasmid (no oily liquid), transfer it to another clean vial, add 20μl TritonX-114, mix at 4-10°C fo...

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Abstract

The present invention provides a method for removing endotoxin from primary pure plasmid or protein. Said method includes the following steps: independently using PMXB or joint-using PMXB and TritonX-114, adding organic solvent, mixing them, making phase separation, or firstly using PMXB and Tritonx-114 and adding organic solvent to make phase separation, the adopting combination method of PMXB gel mixing method and / or PMXB gel chromatography to remove endotoxin from plasmid or protein.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the purification process of plasmids used in gene vaccines and gene therapy and the purification process of proteins, more specifically, to a method for removing endotoxins in primary purified plasmids or proteins. Background technique [0002] Nucleic acid vaccines using "naked engineering plasmids" are the latest or third-generation vaccines that have emerged in the past ten years and are currently being developed. Studies have shown that they are basically ineffective against infectious diseases such as caries, AIDS, The prevention of tuberculosis and malaria and the treatment of hepatitis B have unique advantages and application prospects. The unique advantages of nucleic acid vaccines in inducing the body's cellular immune response make up for the shortcomings of the first and second generation vaccines, and these remarkable advantages are necessary for the prevention and treatmen...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07K1/14C12N15/10
Inventor 刘庆良
Owner SHANGHAI H & G BIOTECH
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