Study On Cloning And Mutation Of Thermostable Direct Hemolysin Gene Of Vibrio Parahaemolyticus

Vp is a major cause of acute gastroenteritis and especially associated with seafood consumption and travlers?diarrhoea . It may cause other diseases such as reactive arthritis and heart diseases. It is classified into two groups by the Kanagawa phenonienon(KP), which is a hemolytic test on Wagatsunia blood agar. More studies revealed that there were most of cases of Vp gastroenteritis caused by KP-positive strain. The molecular biological studies showed that such Vp possesses tdh gene which encoded a protein called thermostable direct haemolysin(TDH). However detailed mechanisms by which TDH display the biological activities remain unclear. This study is to clone and mutate the tdh gene and paid a preliminary foundation of elucidating pathogenesity of Vp, constructing the vaccine candidate. In first part of this study,we design a pair of primers(P l~ P2) , use PCR technique to amplify the special fragment of tdh gene from the genomic DNA, digest it with the restrition endonucleases EcoRJ and BamHl, ligate the digested fragment of tdh gene with pUC 19 digested by the same two restriction endonucleases, transform the ligated liquid into E.coli JM 109. We screen and identify the positive recombinant clone by PCR analysis and restriction analysis. The nucleatide sequence is sequenced by Institute of Military Medicine. Then, we subclone the tdh gene to pTrc99A. The result of SDS-PAGE shows the expressed protein in a highly, soluble and non-fused form. Hemolytic tests show that the protein is active. The expressed protein is purified by the method of superfiltration, gradation sedimentation of ammonium sulfate,and ion-exchange chromatography. The rabbit is immunized on enterocelia by tdh /pTrc99AIJM1O9 which is inactivated by 0.4% formaldehyde at the first ,the fifth, the nineth,the eleventh and the thirteenth day with the dosage at 1.2,1.2,1.8,1.8,1.8 thousand millions.Six days after the last immunization day ,the rabbit serum is abstracted through vein and absorbed by JM I 09.The hemolytic suppression test indicates that there are substance which suppress the hemolyticance of Vp in the rabbit serum. In the second part of this study,we design a mutation primer, construct a pair of mutation primers with the upstream primer of P1, use PCR technique to amplify the special fragment of tdh mutation gene from 1 to 267 base pairQdhm gene 1 -267bp) from the rocombinant plasmid tdhlpUC 19, digest it with the restrition endonuclease EcoRI. Then, we digest the recombinant plasmid tdh/pUC 19 with the restrition endonucleases HincII and BamHl which results the fragment of tdh gene 268-576bp, ligate the above two digested fragment of tdhm gene with pUC 19 digested by the restrition endonucleases EcoRI and BamHI, transform the ligated liquid into E.coli JM 109. We screen and identify the positive recombinant clone by PCR analysis and restriction analysis. The nucleatide sequence is sequenced by the center of Dan-An gene diagnosis of Sun Yat-sen University of Medical Sciences. Then, we subclone the tdhm gene into pTrc99A. the result of SDS- PAGE analysis shows the expressed protein TDHm in a highly, soluble and non-fused form. The result of hemolytic potency shows virulence of the mutant stain lower than that of the wild strain.