Patent application EP15807551A (EP), filed 2015-04-20
Publication number: EP3156487A1
Title: ESCHERICHIA COLI T7 EXPRESSION VECTOR, VECTORS FOR THE CO-EXPRESSION AND CO-PURIFICATION OF RECOMBINANT PEPTIDES IN/WITH CARRIER PROTEINS, USE OF EXPRESSION VECTORS FOR OBTAINING COMPLEXES WITH MULTIPLE ANTIGENS AND IMMUNOMODULATORS
Abstract: The present invention relates to a vector for the expression of recombinant proteins, antigens, pathogen-like particles and immunogenic complexes, said vector (pMRKA vector) being produced by modifying the plasmids containing the gene sequence of the T7 promoter of E. coli, this modification being mainly characterized by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene, and by the insertion of the  par  sequence (partition sequence which determines the efficient segregation of the plasmids in daughter cells during cell division). Also provided are expression vectors based on the pMRKA plasmid, which additionally comprise at least one of the gene sequences of the exosome of P. abyssi, which vectors are designated pMRKA-EXO, pMRKA-RING and pSUMAC. The invention also provides the vectors additionally comprising gene sequences with immunomodulatory or immunoregulatory activity, preferably the pMRKA-Z-Z-EXO and pMRKA-Z-Z-RING vectors. Other aspects of the invention include the method for producing said expression vectors and the use of the obtained vectors.
IPC codes: A61K  39/    00            A I
A61K  39/   385            A I
A61P  37/    04            A I
C12N  15/    62            A I
C12N  15/    68            A I
C12N  15/    69            A I
C12N  15/    70            A I
Claims: number: 1
text: Escherichia coli T7 expression vectorcharacterized by : (a) having a high number of copies per cell; (b) being highly stable in high density cellular fermentation conditions; (c) sized about 2 Kpb to 4kpb; (d) having an antibiotic resistant marker.
number: 2
text: Vector of claim 1,characterized by having 300 to 500 copies per cell.
number: 3
text: Vector of claim 1,characterized in that the stability in high density cellular fermentation conditions is provided by the presence of apar sequence of pSC101 plasmid.
number: 4
text: Vector of claim 1,characterized in that thepar sequence comprises SEQ ID No: 11.
number: 5
text: Vector of claim 1,characterized in that said antibiotic resistant marker is the kanamycin resistant gene.
number: 6
text: Vector of any previous claims,characterized in that pMRKA plasmid comprises an nucleotide sequence SEQ ID No: 19.
number: 7
text: Vector for polypeptides expression of fusion to carrier proteins,characterized in that it comprises: (a) a vector according to any of claims 1 to 6; and (b) at least one P. abyssi exosome gene encoding at least one immunogen proteins, antigens or immunoregulatory molecules-carrier protein.
number: 8
text: Vector of claim 7,characterized in that it comprises P. abyssi exosome gene encoding at least one of Rrp4, Rrp41 and Rrp42 proteins.
number: 9
text: Vector of claim 8,characterized in that it comprises P. abyssi exosome genes encoding at least two of the Rrp4, Rrp41 and Rrp42 proteins.
number: 10
text: Vector of claim 8,characterized in that it comprises P. abyssi exosome genes encoding three of the Rrp4, Rrp41 and Rrp42 proteins.
number: 11
text: Vector of claim 8,characterized in that the vector is pMRKA-EXO and it comprises the nucleotide sequence SEQ ID No: 36.
number: 12
text: Vector of claim 9,characterized in that it comprises P. abyssi exosome genes encoding Rrp41 and Rrp42 proteins.
number: 13
text: Vector of claim 10,characterized in that the vector is pMRKA-RING plasmid and it comprises the nucleotide sequence SEQ ID No: 37.
number: 14
text: Vector of claim 8,characterized in that it comprises P. abyssi exosome genes encoding Rrp42 protein.
number: 15
text: Vector of claim 14,characterized in that the vector is pSUMAC plasmid and it comprises the nucleotide sequence SEQ ID No: 40.
number: 16
text: Vector of any of the preceding claims,characterized in that additionally comprises at least one immunoregulatory gene sequence.
number: 17
text: Vector of claim 16,characterized in that said at least one immunoregulatory gene sequence is the Z domain sequence from A. protein of S. aureus.
number: 18
text: Vector of claim 17,characterized in that said Z domain sequence from A protein of S. aureus is a Z-Z nucleotide sequence that comprises the SEQ ID No: 41 encoding protein of SEQ ID No: 42.
number: 19
text: Vector of claim 18,characterized in that said Z-Z nucleotide sequence comprises the SEQ ID No: 41 and it is in-fusion cloned in the encoding region of N-terminal end of said Rrp42 composed in pMRKA-EXO plasmid, resulting in pMRKA-ZZ-EXO vector that comprises the nucleotide sequence SEQ ID No: 43.
number: 20
text: Vector of claim 18,characterized in that said Z-Z nucleotide sequence comprises the SEQ ID No: 41 and it is in-fusion cloned in the encoding region of N-terminal end of said Rrp42 comprised in pMRKA-RING plasmid, resulting in pMRKA-ZZ-RING vector that comprises the nucleotide sequence SEQ ID No: 45.
number: 21
text: Method for obtaining an expression vector as defined in claims 1 to 6,characterized in that it comprises the steps: (i) exchange the ampicillin resistant gene in vector of SEQ ID NO: 1 with the kanamycin resistant gene; (ii) amplification of the plasmid fragment of SEQ ID NO: 1 corresponding to the segment located among nucleotides 804 to 1857; (iii) binding the amplified segments from step (ii) with the kanamycin resistant gene; (iv) amplification of the plasmid obtained in step (iii) to insert restriction sites in base 2607 of pMK plasmid; (v) amplification ofpar sequence and insertion of the amplified sequence resulting among the restriction sites located at the base 2607 from pMK for obtaining pMRK plasmid; and (vi) eliminating the restriction sites of BgIII and Ncol in the kanamycin resistant gene in pMRK vector for obtaining pMRKA plasmid.
number: 22
text: Method of claim 21,characterized in that said resistant gene exchange of step (i) is made by amplifying the segment obtained from primers of SEQ ID No: 2 and SEQ ID No: 3, and the amplified segment comprises SEQ ID No: 4.
number: 23
text: Method of claim 21,characterized in that said amplification step (ii) is executed from primers of SEQ ID No: 6 and SEQ ID No: 7.
number: 24
text: Method of claim 21,characterized in that said binding step (iii) results in pMK plasmid that comprises SEQ ID No: 8.
number: 25
text: Method of claim 21,characterized in that saidpar sequence amplification of step (iv) is executed from primers of SEQ ID No: 9 and SEQ ID No: 10, resulting in the amplified sequence SEQ ID No: 11.
number: 26
text: Method according to any of the claims 21 to 25,characterized in that said pMRK plasmid is obtained by amplifying pMK plasmid, from the primers SEQ ID No: 12 and SEQ ID No: 13, and insertion into SEQ ID No: 11, resulting in the plasmid of SEQ ID No: 14.
number: 27
text: Method of claim 21,characterized in that said elimination of BgIII and Ncol restriction sites in kanamycin resistant gene of step (vi) is executed by site-directed mutagenesis, and the elimination of said BgIII restriction site is executed using primers of SEQ ID No: 15 and SEQ ID No: 16, and the elimination of said Ncol restriction site is executed using primers of SEQ ID No: 17 and SEQ ID No: 18.
number: 28
text: Method, according to any of claims 21 to 27,characterized in that said pMRKA plasmid comprises the SEQ ID No: 19.
number: 29
text: Method for obtaining an expression vector as defined in claims 7 to 15,characterized in that it comprises the steps:
number: 30
text: Method of claim 29,characterized in that said cloning in step (iv) is executed between Xbal and Hindlll sites of pMRKA plasmid.
number: 31
text: Method, according to any of the claims 29 and 30,characterized in that said at least one P. abyssi exosome gene sequence of step (ii) comprises gene sequences encoding Rrp4, Rrp41 and Rrp42 proteins of P. abyssi exosome.
number: 32
text: Method of claim 31,characterized in that there are additionally provided sequences SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24 and SEQ ID No: 25 to allow in-fusion cloning of said gene sequences encoding Rrp4, Rrp41 and Rrp42 proteins of P. abyssi exosome.
number: 33
text: Method of claim 31,characterized in that said gene sequences comprises SEQ ID No: 26, SEQ ID No: 28 and SEQ ID No: 30 encoding Rrp4, Rrp41 and Rrp42 proteins, respectively, of P. abyssi exosome.
number: 34
text: Method, according to any of claims 29 to 32,characterized in that P. abyssi exosome gene sequences are gathered in the order SEQ ID No: 22-SEQ ID No: 26-SEQ ID No: 23- SEQ ID No: 28-SEQ ID No: 24-SEQ ID No: 30-SEQ ID No: 25.
number: 35
text: Method of claim 34,characterized in that the gathering of said gene sequences forms SEQ ID No: 32.
number: 36
text: Method of claim 35,characterized in that it is additionally synthetized a double-stranded DNA comprising SEQ ID No: 32 that is cloned in pMRKA plasmid, between Xbal and Hindlll plasmid, to form SEQ ID No: 36 comprised in pMRKA-EXO plasmid.
number: 37
text: Method, according to any of the claims 29 and 30,characterized in that said at least one P. abyssi exosome gene sequence of step (ii) comprises gene sequences encoding Rrp41 and Rrp42 proteins of P. abyssi exosome.
number: 38
text: Method of claim 37,characterized in that said gene sequences comprises the sequences SEQ ID No: 28 and SEQ ID No: 30 respectively encoding Rrp41 and Rrp42 proteins of P. abyssi exosome.
number: 39
text: Method of claim 38,characterized in that said P. abyssi exosome gene sequences were amplified from pMRKA-EXO plasmid to form SEQ ID No: 37 comprised in pMRKA-RING plasmid.
number: 40
text: Method, according to any of the claims 29 and 30,characterized in that said at least one P. abyssi exosome gene sequence of step (ii) comprises gene sequences encoding Rrp42 protein of P. abyssi exosome.
number: 41
text: Method of claim 40,characterized in that said sequence encoding Rrp42 protein of P. abyssi exosome was amplified from pMRKA-EXO plasmid, using oligonucleotides with SEQ ID No: 38 and SEQ ID No: 39
number: 42
text: Method of claim 41,characterized in that the DNA fragment resulting from the amplification of sequences SEQ ID No: 38 and SEQ ID No: 39 was cloned in pMRKA vector to form SEQ ID No: 40 comprised in pSUMAC plasmid.
number: 43
text: Method, according to any of claims 21 to 42,characterized in that it additionally includes in pMRKA-EXO and pMRKA-RING plasmids at least on immunoregulatory gene sequence.
number: 44
text: Method of claim 43,characterized in that said at least one immunoregulatory gene sequence is the Z domain sequence from A protein of S. aureus for tandem expression of two Z domain, Z-Z domains copies, that is in-fusion cloned with the region encoding N-terminal end of Rrp42 protein comprised in pMRKA-EXO and pMRKA-RING vectors
number: 45
text: Method of claim 44,characterized in that said gene sequence encoding Z-Z domains is in-fusion cloned with the region encoding the N-terminal end of Rrp42 protein among Ncol and Sail sites in pMRKA-EXO e pMRKA-RING plasmids.
number: 46
text: Method of claim 45,characterized in that pMRKA-ZZ-EXO vector is produced and it comprises SEQ ID No: 43.
number: 47
text: Method of claim 45,characterized in that pMRKA-ZZ-RING vector is produced and it comprises the SEQ ID No: 45
number: 48
text: Usage of the expression vectors defined in claims 1 to 6,characterized by being in expression of recombinant proteins, antigens and immunogenic complexes.
number: 49
text: Usage of claim 48,characterized in that the vector is the pMRKA plasmid.
number: 50
text: Usage of the expression vectors defined in claims 7 to 15,characterized by being in expression of recombinant proteins, antigens and immunogenic complexes fused to the carrier proteins.
number: 51
text: Usage of claim 50,characterized in that said carrier proteins are P. abyssi exosome proteins, preferably Rrp4, Rrp41 and Rrp42 proteins..
number: 52
text: Usage of claim 51,characterized in that the vector is the plasmid selected from pMRKA-EXO and pMRKA-RING, said vector being especially usable in the expression and co-purification of recombinant proteins to form pathogen like particles.
number: 53
text: Usage of claim 50,characterized in that the vector is the pSUMAC plasmid that is especially usable in obtaining carrier proteins of peptide vaccines.
number: 54
text: Usage of the expression vectors defined in claims 16 to 20,characterized by being in expression of recombinant proteins, antigens and immunogenic complexes fused to the carrier proteins and additionally having immunomodulatory activity.
Office action: search_report, dated 2017-04-19
Rejection reasons: inventive_step_epc_56
Prior art cited: ref_id: WO0181580
ref_type: patent
severity: obviousness
ref_id: EP1609867
ref_type: patent
severity: obviousness
ref_id: US2008274137
ref_type: patent
severity: obviousness
ref_id: WO2010101750
ref_type: patent
severity: obviousness
ref_id: WO2013022358
ref_type: patent
severity: obviousness
ref_id: WO2013024114
ref_type: patent
severity: obviousness
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ref_type: npl
severity: obviousness
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ref_type: npl
severity: obviousness
Final disposition: pending (2017-04-19)