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Production of recombinant anthrax protective antigen and its special expression plasmid

A protective antigen and expression plasmid technology, applied in the field of the preparation of recombinant anthrax protective antigen, can solve the problems of affecting the protein to form a natural conformation, difficult to put into practical application, difficult to apply, etc., and achieve good biological activity and immunogenicity. Applied value, effect of profound theoretical significance

Active Publication Date: 2007-09-12
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is innovative to use lactic acid bacteria or Salmonella to express PA to develop oral vaccines, it is difficult to put into practical application in the short term
The pQE30 vector system was used to express rPA in Escherichia coli. Although the yield was high, the recombinant protein formed inclusion bodies, which required denaturation and renaturation steps during the purification process, which may affect the natural conformation of the protein; 6×His tags, which will bring difficulties to future applications, because the components of human vaccines should preferably not contain foreign tag sequences
There are also authors who secrete and express rPA in Escherichia coli, but the expression level is low, and only about 3mg rPA can be obtained from 1L culture

Method used

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  • Production of recombinant anthrax protective antigen and its special expression plasmid
  • Production of recombinant anthrax protective antigen and its special expression plasmid
  • Production of recombinant anthrax protective antigen and its special expression plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1, the construction of expression plasmid pAS-PA

[0016] 1. Construction of expression vector pAS20

[0017] Using the general expression vector pASK84 (Skerra A.A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 1994, 141: 79-84.) as a template, it was separated from the general cloning vector pUC18 Digest with EcoR I / HindIII, recover the vector fragment of pASK84 and the multiple cloning site fragment of pUC18 by agarose gel electrophoresis, connect the two, transform Escherichia coli XL1-blue by TSS method, and extract the plasmid with EcoR I / HindIII enzyme digestion identified, and the sequence of the plasmid with the correct insertion of the multiple cloning site was determined, and the plasmid with the correct sequence was named pAS18. pAS18 was double digested with BamH I / HindIII, the vector fragment was recovered by electrophoresis, blunted with Klenow enzyme and ligated, transf...

Embodiment 2

[0026] Embodiment 2, expression and purification of recombinant anthrax protective antigen (rPA)

[0027] 1. Express

[0028] Pick a single colony of E.coli DH5α engineered bacteria transformed with plasmid pAS-PA from solid LB medium, insert it into LB medium containing 100 μg / mL ampicillin, and cultivate it to OD at 37°C and 250 rpm 600 = 0.7-0.8, add IPTG to 0.5mmol / L, induce expression at 28°C at 250 rpm for 5h, and collect the bacterial pellet by centrifugation.

[0029] 2. Isolation of bacterial periplasm

[0030] 11 Induced culture cells after centrifugation were resuspended with 100mL 20% sucrose solution (20mmol / L TrispH8.0, 1mmol / LEDTA, 1mmol / L PMSF), placed on ice for 5min, and centrifuged at 8000g for 20min at 4°C. 100mL 5mmol / L MgSO for precipitation 4 (1mmol / L PMSF), placed on ice for 10min, centrifuged at 10000g for 10min at 4°C, and the supernatant was collected for purifying rPA.

[0031] 3. Ion exchange

[0032] The supernatant collected in step 2 was fi...

Embodiment 3

[0041] Example 3, biological activity and immunogenicity analysis of rPA prepared by the method of the present invention

[0042] 1. Bioactivity analysis of rPA

[0043] The combination of anthrax protective antigen (PA) and lethal factor (LF) is called lethal toxin (LT), which can lead to the death of sensitive cells (such as mouse macrophage J774A.1) in vitro.

[0044] Mouse macrophage J774A.1 was treated with 10 5 / ml inoculated in a 96-well culture plate, grown to 90% full, different concentrations of rPA and recombinant LF (rLF, see Gupta P, et al. Expression and purification of the recombinant lethal factor of Bacillus anthracis. Infection and Immunity1998, 66 (2): 862-865.) were added to the cell wells, incubated at 37°C for 3 hours, and the cell viability was observed by the MTT method. The results are shown in Figure 4. When one of rPA and rLF was fixed and the concentration of the other protein was increased, the cell survival rate decreased significantly; while on...

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Abstract

The invention provides a method and the expressed plasmid, which prepare the recombined anthrax protective antigen. The DNA sequence of the plasmid is the sequence 1 or the sequence which can cross with the sequence 1.The method gets the engineering strain by translating the E.coli using the expressed plasmid of the recombined anthrax protective antigen, so next to ferment the strain and get the metabolite. The method can get the more rPA, which have the true structure and avoid degrading by the proteinase. So we can get the 15mg / L rPA that's purity is above 95%, and the N terminal is accordance with the nature antigen.

Description

technical field [0001] The invention relates to a method for preparing recombinant protein and its special expression plasmid, in particular to a method for preparing recombinant anthrax protective antigen and its special expression plasmid. Background technique [0002] Anthrax toxin includes three protein factors, namely protective antigen (PA), lethal factor (LF) and edema factor (EF), among which PA can induce the body's protective immunity and is currently approved by the US Food and Drug Administration (FDA) ) is the main immunoactive component of the approved anthrax vaccine (AVA). At present, the production of AVA still uses the traditional process, using Al(OH) 3 PA is adsorbed from the culture of the attenuated strain of anthrax, so it contains trace amounts of other components, such as LF, EF, etc. The side effects such as local pain and edema during AVA use may be related to this. In addition, the AVA immunization procedure is also relatively cumbersome, requir...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02C12N1/21C07K14/245C12R1/19
Inventor 陈薇徐俊杰董大勇付玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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