Efficient and Soluble Expression of α Protein of Clostridium perfringens and Preparation

Animal Husbandry and Feed Science, 2017, 9(5): 284 -288, 305DOI : 10.19578/j. cnki. ahfs.2017.05.007

Bfici^nt and Soluble Expression of a Protein of Clos- fr/d/i/m perfr/ngens and Preparation of GeneticEng^ neering Subunit Vaccine

Sun Yu , Yang Lin , W ang Chuanbin , Dong Hao , Qu Ping , Zhao Bolin , Hu Dongmei , Yang Tianyi , Song Xiaohui"

China Animal Disease Control Center, Beijing 102600, China

Abstract

[Objective] The paper w as to develop genetic engneering vaccine that can express a exotoxin antigen protein efficiently without destroying its immuno-genicity for preventing and controlling the diseases caused by Clostridium pejfringens. [Method] Efficiently expressed soluble rec from Escherichia coli expression system by optimizing codon , removing signal peptide , selecting sequences with better hydrophilicity and antigenicity , and optimizing expression conditions. [Result] Mice obtained higher serum antibody level when immunized by a protein , and the immune protection rates a ainst type A , type B , type C and type D C . pejfringegs were 100% , 90% , 85% and 90% , respectively. The antibody titer of mice within 7 -14 d after the third immunization reached the peak. [ Conclusion ] The a protein h as good immunogenicity , and can be further used to develop genetic engineering subunit vaccines for preventing C. perfrin-

ge?g.

Key words COstridium pejfringens ; a protein ; Soluble expression and purification ; Genetic engineering subunit vaccine

Clostridium efringens is the common clostridium causing gas

gangrene in clinical. It produces large amounts of gas by decompo­sing sugars in muscles and connective tissues , and leads to severe emphysema of tissue , which further affects blood supply , causing large area necrosis of animal tissue[1]. Gas gangrene of ruminants , enterotoxaemia , hemorrhagic enteritis , sudden death syndrome of

sheep and cattle , lamb dysentery are caused by

C. perfringens^22.

a toxin is the most important exotoxin in all toxin genes of C. per- fnngegs , and type A , B , C , D and E bacteria can produce the toxin[ 52. The gene plc coding a toxin locates on chromosome ,

with the size of 1 194 bp and the molecular weight of 42. 5 Ku , which can encode 398 amino acids , and mature peptide and signal peptide consist of 370 and 28 amino acids , respectively. Current­ly ,scholars at home and abroad focus on function and pathogene­sis of a toxin gene. a toxin hydrolyzes membrane phospholipid of cell membrane relying on sphingomyelinase and phospholipase C , which further destroys cell membrane structure and leads to rapid pyrolysis and death of cells ; meantime , the toxin is sensitive to pancreatin , and w ill easily lose activity after contact6-8]. a toxin gene is relatively conservative , although there are differences in 1.3% nucleotide and 1.2% amino acids sequence on average among different strains , different nucleotides and encoding amino acids do not affect the activity of a toxin its e l[ -2]. The promoter

of a toxin gene can be recognized by RNA polymerase of Esche­

richia coli , so a toxin can be efficiently expressed in both C. per-fringegs and E. coli under the action of its promoter. Developing

genetic engineering subunit vaccine that can express a exotoxin antigen protein without destroying its immunogeeicity and can pre­

Received : May 15 , 2017 Accepted : June 27 , 2017

Supported by the 13th Five-Year National Key Research and Development Pro­

gram (2016YFD0500901).

"Corresponding author. E-mail : syl9830908@ 163. com

vent and control the diseases caused by a exotoxin of

C. perfrii-

gegs is a technical problem urgently to be solved[13].

1 Materials and Methods

1.1 Materials

pET30a fusion expression vector was purchased

from Novagen company ; BL21 ( D E 3) competent cells were brought from Beijing TmnsGen Biotech Co. , Ltd. Restriction en­

zymes

BamH I , Xho I , T4 DNA ligase , 2 000 DNA Marker , SDS ,

IPTG , T 〇a PCR Master Mix were received from TaKaRa ( Dalian)

Engineering Co. , Ltd. Agarose , DNA Extraction K it , DNA rapid purification k it , plasmid rapid extraction k it were got from Beijing TransGen Biotech Co. , Ltd. Pre-dyed protein Marker was the product of Fermentas Cooperation. Protein purification column (nickel column 5 m L) , molecular sieve (Superdex 2000) were purchased from GE Company. HRP-labeled sheep anti mouse IgG , Freund’s complete adjuvant and incomplete adjuvant were manufactured by Sigma Company. Target gene sequence of fusion protein was synthesized by BGI Biological Technology Co. , Ltd.

C. perfringem including type A C57-10 ,

type B C58-5 , type C

C59-4 and type D C60-11 were products of China Institute of Vet-

erinam Drug Control.

1.2 Methods

1.2.1 Synthesis and primer design of a recombinant gene. a re­

combinant gene was designed by removing protein signal peptide and optimizing codon sequence. A pair of primers was designed according to the sequence of a recombinant gene , while

BamH I

and Xho I restriction sites wereadded at 5’ and 3’ ends of primers respectively , for amplification of target fragment ( underlined parts are restriction sites).

Forward primer F : 5,-GGATCCATGTTTTGGGACCCGGA-

CACCGAC-3,;Reverse primer R : 5,-CTCGAGTTATTTGATGTTATAGGT-

GCTGT-3’.

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