Patent Application: US-51050906-A

Abstract:
a nucleic acid sequence encoding all or part of an 18 - 19 kda helicobater pylori protein is described to which immunoreactivity is detected in h . pylori negative individuals . a process for the production of a recombinant form of this protein and its use , particularly as a vaccine to provide immunological protection against h . pylori infection are also described .

Description:
we have generated dna sequence information identifying the 18 - 19 kda protein as a bacterioferritin . we have also generated a recombinant 18 kda protein and expressed this in e . coli . this recombinant protein was found to be recognized immunologically by antisera from individuals positive for antibody to the 18 kda helicobacter bacterioferritin . this recombinant protein will form the basis for a putative vaccine for h . pylori . fig1 is a western blot analysis of the recombinant 18 kda protein expressed in e . coli . cloning and expression of the helicobacter pylori 18 kda gene . deoxyribonucleic acid ( dna ) was extracted from helicobacter pylori as described by silhavy et al . *( 1984 ). oligonucleotides ( or “ primers ”) specific for the 5 ′ and 3 ′ termini of the 18 kda gene were generated . the forward or 5 ′ oligonucleotide ( designated hp18cf ) was modified to incorporate an nde 1 restriction endonuclease site . additional modifications were made to increase the stability of the binding of the oligonucleotide to its target sequence and to prevent intramolecular secondary structure . the sequence of the hp18cf oligonucleotide is ( from 5 ′ to 3 ′): the reverse or 3 ′ oligonucleotide ( designated hp18cr ) was extensively modified to incorporate a bamh1 restriction endonuclease site and a 5 ′ tail . the 15 3 ′ nucleotides of this oligonucleotide correspond to the helicobacter pylori 18 kda gene sequence . the sequence of the hp18cr oligonucleotide is ( from 5 ′ to 3 ′): these oligonucleotides were used in a polymerase chain reaction ( pcr ) to amplify the helicobacter pylori 18 kda gene sequence . the reaction conditions were as follows : between 50 and 100 ng of helicobacter pylori dna was added to 75 pool of each primer , 0 . 4 mm of each deoxyribonucleotide triphosphate ( dntp ), a final concentration of 4 mm mgso 4 , 1 fold ‘ thermopol ’ ( new england biolabs ) reaction buffer ( composition : 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 20 mm tris - hcl ( ph 8 . 8 at 25 degrees c . ), 2 mm mgso 4 , 0 . 1 % triton x - 100 ), and deionized water was added to bring the reaction volume to 50 ul . the reaction mixture was overlaid with 50 ul paraffin oil and placed in a perkin - elmer thermocycle at 90 degrees c . 1 unit vent , dna polymerase ( new england biolabs ) was then added . a ‘ touchdown ’ pcr procedure was utilized ( don et al . 1989 )*. the cycling conditions were as follows : the dna was denatured at 94 degrees c . for 2 . 5 minutes . this was followed by 2 cycles of 94 degrees for 30 seconds ( denaturation step ), 65 degrees for 50 seconds ( annealing step ), and 72 degrees c . for 20 seconds ( extension step ). this was followed by 2 cycles of the same conditions , with the exception that the annealing temperature was dropped 5 to 64 degrees c . after 2 cycles at 64 degrees c ., the annealing temperature was reduced to 63 degrees c . for a further 2 cycles , and this pattern was followed until the annealing temperature was reduced to 60 degrees c . for 28 cycles . the reaction products were purified on a 4 % low melting point agarose gel ( nusieve gtg ; fmc bioproducts ). the dna fragment was excised from the gel and the agarose was digested using β - agarase 1 ( new england biolabs ) and the dna recovered following precipitation with isopropanol , according to the manufacturer &# 39 ; s supplied protocol . the purified dna fragment corresponding to the 18 kda protein coding gene was then digested with the restriction enzymes nde 1 and bamn1 , ( boehringer mannheim ), each of which occurs only once on the amplified fragment . 10 units of each enzyme was added to approximately 3 ug of dna in a final concentration of 1 × the manufacturer &# 39 ; s supplied restriction buffer b in a 40 ul reaction volume . the reaction mix was incubated at 37 degrees c . for 3 . 5 hours . the expression vector used was pet16b ( novagen ). the 1 . 6 ug of the vector was digested using nde1 and bamh 1 under the same conditions as described for the amplified fragment . the resulting 5 ′ phosphate groups were removed using calf intestinal alkaline phosphatase ( ciap : newengland biolabs ) according to the manufacturer &# 39 ; s instructions . the enzyme was inactivated by incubating the reaction mixture in the presence of 5 mm edta at 65 degrees c . for 1 hour followed by a phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) extraction , followed by a chloroform / isoamyl alcohol ( 24 : 1 ) extraction . both the digested fragment and the digested vector were gel purified on a 3 % low melting point agarose gel . ( nusieve gtg ; fmc bioproducts ), and . the agarose was digested using β - agarase 1 ( new england biolabs ), according to the manufacturer &# 39 ; s instructions . the dna fragments were allowed to remain in the resultant reaction mixture without further purification . the amplified fragment was then ligated to the vector dna as follows . approximately 200 ng of vector was ligated to approximately 100 ng of the insert dna in 1 × ligation reaction buffer and 3 units of t4 dna ligase ( boehringer mannheim ) in a reaction volume of 30 ul at 20 degrees c . for 16 hours . the products of this reaction were used to transform competent e . coli xl 1 - blue cells ( bullock at al . 1987 ) using a standard cacl 2 transformation procedure ( sambrook , et al ., 1989 ). transformed xl1 - blue cells were selected on lb medium ( sambrook et al ., 1989 ) supplemented with 50 ug per ml ampicillin and grown at 37 degrees c . suitable colonies were picked and used to inoculate 10 ml lb broth supplemented with 50 ug per ml ampicillin and grown with shaking at 37 degrees c . the plasmids were purified from these cultures using a standard alkaline lysis plasmid preparation procedure ( sambrook , et al ., 1989 ), and an aliquot digested with nde 1 and kindzz7 according to the manufacturers instructions ( boehringer mannheim ) to verify the presence of the insert as compared to a size standard and pet16b without an insert . two plasmids shown to have the appropriate insert ( designated pet16b - 18 . 1 and pet16b - 18 . 2 ) were then used to transform the e . coli expression hosts bl21 de3 ( studier and moffat , 1986 ) and novablue de3 ( novagen ) using a standard cacl 2 transformation procedure ( sambrook , et al ., 1989 ●) supplemented with 50 ug per ml ampicillin ( novablue de3 ) or 50 ug per ml ampicillin and 34 ug per ml chloramphenicol ( sl21 de3 ) and grown at 37 degrees c . transformed cells were selected by plating on solid lb medium . a colony of each host representing each plasmid isolate was picked after 16 hours incubation and used to inoculate 50 ml lb broth supplemented with antibiotics as described above and grown until the optical density at 600 nm was approximately 0 . 6 . the expression of the 18 kda protein from the expression vector was then induced by the addition of isopropyl b - d - thiogalactopyranoside ( iptg ) to 15 a final concentration of 1 mm and incubation was continued for a further 2 . 5 hours at 37 degrees c . with shaking . the cells were then harvested by centrifugation at 4000 × g for 10 minutes and resuspended in 12 ml of 50 mm tris - hcl ( ph 8 . 0 at 25 degrees c .) followed by a further centrifugation at 4000 × g for 10 minutes . the purified dna fragment corresponding to the 18 kda protein was sequenced using forward and reverse universal sequencing primers . the dna was sequenced in the forward and reverse orientations . sequencing was performed using an abi automated sequencer and a genpak pcr based fluorescent dideoxy chain terminator termini sequencing kit . the sequence of bases between the termini of the internal pcr primers is : two transformed e . coli expression hosts ( bl21 de3 and novablue de3 ) were subjected to sds - page ( 12 . 5 % t ) followed by western blotting analysis . the western blots were probed with serum obtained from children uninfected with h . pylori and developed by enhanced chemiluminescence . as illustrated in fig1 , two of the three sera recognised the recombinant 18 kda protein after induction of expression of the protein with iptg . in addition , the three sera recognised a number of e . coli proteins . it is understood that the recombinant proteinaceous material of the invention is used as a vaccine against h . pylori infection , and in particular as a therapeutic immunogen for eradication of h . pylori infection . the vaccine may include the proteinaceous material according to the invention in combination with other components such as a pharmaceutically acceptable carrier , a suitable adjuvant such as interleukin 12 or a heat shock protein , an antibiotic and / or an antibacterial agent such as bismuth salts . the vaccine may be administered in a number of different ways , namely , orally , intranasally , intravenously or intramuscularly . the invention is not limited to the embodiments hereinbefore described which may be varied in detail . marshall , b . j . and warren , j . r . 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