Expression And Genetic Stability Analysis Of α-β2-ε-β1 Fusion Toxin Genes Of Clostridium Perfringens In Lactobacillus Casei

Clostridium perfringens is a Gram-positive rod, which is widely distributed in nature. It is the etiological agent of several human and animal diseases. It can cause alimentary toxicosis, traumatic gas gangrene, enterotoxaemia and enteritis necroticans. Exotoxin is the main pathogenic factor of Clostridium perfringens, including the alpha, beta, epsilon and ι toxins, among of them alpha, beta, epsilon toxins are common. Based on the harmfulness and the characteristics of intestinal infection of Clostridium perfringens, lactobacillus as live carrier vaccine passing the immune antigen, EGFP as a marker, antibiotic selection markers and constitutive recombinant lactobacillus expression system was bulid to express fusion toxin α, β2, ε, β1 gene of Clostridium perfringens, for providing material foundation to develop mucosal immune agents of Clostridium perfringens.In this study α-β2-ε-β1 gene was inserted into constitutive expression vector p PG-T7g10-PPT and recombinant lactobacillus casei plasmid p PG-α-β2-ε-β1 was constructed. Then EGFP gene was inserted before α-β2-ε-β1 gene in pPG-α-β2-ε-β1 by Sac I restriction enzyme cutting site and formed the recombinant Lactobacillus casei plasmid pPG-E-α-β2-ε-β1. The plasmid pPG-E-α-β2-ε-β1 was transformed into lactobacillus casei by electricity transformation to obtain recombinant bacteria pPG-E-α-β2-ε-β1/L.casei 393, which was found α-β2-ε-β1 shorten when it was generated 3~4th generations. According the result of PCR, restriction enzyme digestion and sequencing, it can be concluded that α-β2-ε-β1 has become into α-β1 and β2, ε gene was lost during generation of recombinant Lactobacillus casei. When recombinant Lactobacillus casei was generated 50 th generations, the α-β1 gene was found stable inheriting. When the recombinant bacteria p PG-E-α-β2-ε-β1/TG1 was generated 50 th generations, the generations was stable inheritance, which illustrated the gene shorten phenomenon relative to the host bacterium.The linker sequences(GPGPYV) in α-β2-ε-β1 gene were same, which was regarded as repetitive sequence and considered relative to the gene shorten phenomenon. In order to verify this hypothesis, α-β2-ε-β1 gene was reconstructed by different sequence linker and it was inserted in plasmid p PG-T7g10-PPT after the EGFP gene, which formed the new plasmid p PG-E-α-β2-ε-β1’. The plasmid p PG-E-α-β2-ε-β1 was transformed into Lactobacillus casei by electricity transformation to obtain recombinant bacteria p PG-E-α-β2-ε-β1’/L. casei 393, wich was found stable inheriting when it was generated 50 th. The fusion protein was detected by the laser confocal microscope, flow cytometry and Western blot test. The size of this fusion protein about 196 KDa according the result of Western blot. The results of laser confocal microscope and flow cytometry revealed that fusion protein can stable expressed in the recombinant lactobacillus.In conclusion, the result is meaningful that several same sequence linkers induce the instability of fusion gene, it has directive significance to construction of lactobacillus expression system and recombinant living vectored vaccines. Meanwhile, construction of recombinant lactobacillus expression system with EGFP selection markers system laid the foundation for the development of polyvalent subunit vaccine of Clostridium perfringens.