# Patent

## Title
Human long pentraxin 3 expression system and uses thereof

## Abstract

The present invention relates to an eukaryotic expression vector comprising a nucleotide sequence encoding for the human long pentraxin PTX3 protein under the control of an effective promoter and a nucleotide sequence encoding for a selectable marker, recombinant human cell able to provide expression of proteins encoded by the vector and method for the production of the human long pentraxin PTX3 protein.

## Background
This application is a U.S. national stage of PCT/EP2010/060469 filed on Jul. 20, 2010 which claims priority to and the benefit of European Application No. 09166759.2 filed on Jul. 29, 2009, the contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to human derived cellular system able to express high levels of the human pentraxin 3 (hPTX3) protein, methods and material used.
BACKGROUND TO THE INVENTION
Human long pentraxin 3, PTX3 or hPTX3 (GENBANK ® Accession Number BD 131701) is a multimeric glycoprotein composed of eight subunits linked by disulphide bridges.
The authors of the present invention have already designed an expression system for the production of PTX3 in HEK293F human cell line. This system is based on a plasmid containing the neomycin resistance gene wherein the PTX3 gene is under the control of a human Ubiquitin C promoter sequence. Such system avoids the potential formation of a chimeric PTX3 derived from endogenous production of PTX3 by a cell line of non-human origin. The best producer isolated clone, named 2F12, was selected among those obtained. A production of about 20 mg/L of PTX3 was obtained, which was not sufficient for commercial production needs.
With the aim to increase PTX3 expression levels, the authors of the present invention have constructed a plasmid in which the PTX3 gene was under CMV promoter control. The plasmid was used to re-transfect the PTX3 expressing clone 2F12. The productivity level detected in the new isolated transfectomas was higher that expected, around 80 mg/L.
DESCRIPTION OF THE INVENTION
A clone of human origin expressing high levels of human PTX3 was obtained using an experimental strategy, including the following steps:

    
    
        a) construction of plasmid expression cassettes carrying the human PTX3 under the control of CMV promoter;
        b) insertion of the hygromycin resistance cassette in said plasmid, in order to select stable transfectomas originating from a re-transfection of PTX3 expressing clone G418 resistant;
        c) verifying the identity and the levels of expressed recombinant proteins;
        d) biochemical characterization of the recombinant hPTX3.
    
    


The content of PCT/EP2009/050937 is hereby incorporated by reference.
It is an object of the invention an eukaryotic expression vector comprising a nucleotide sequence encoding for the human long pentraxin PTX3protein under the control of an effective promoter and a nucleotide sequence encoding for a selectable marker, having essentially the sequence of SEQ ID NO: 1.
The vector is preferentially use to transform host cell, preferably wherein the host cell is a recombinant human cell already able to express the human long pentraxin PTX3 protein, more preferably wherein the recombinant human cell is the recombinant 293F/PTX3/2F12 clone deposited at ECACC with no. 08011001. In a particular aspect the vector is linearized.
It is another object of the invention a recombinant cell able to express the human long pentraxin PTX3 protein encoded by the vector as above disclosed, preferably it is recombinant HEK293F cell line, more preferably it is the recombinant MS24PTX clone deposited at Health Protection Agency, Culture Collections Centre For Emergency Preparedness and Response Salisbury UK, with no. 09072902.
It is another aspect of the invention the use of the recombinant cell as above disclosed for the production of human long pentraxin PTX3 protein.
It is another object of the invention a process for the production of the recombinant human long pentraxin PTX3 protein comprising:

    
    
        a) transfecting a recombinant human cell already expressing recombinant human long pentraxin PTX3 protein, with a selectable plasmid in which the human long pentraxin gene is under the control of the CMV promoter;
        b) selecting and growing transfected recombinant human cell;
        c) purifying the human long pentraxin PTX3 protein from the culture medium of the transfected recombinant human cell.
    
    


Preferably the recombinant human cell expressing a recombinant human long pentraxin PTX3 protein is a recombinant HEK293F cell line, more preferably it is the recombinant 293F/PTX3/2F12 clone deposited at ECACC under number 08011001.
In a preferred embodiment the purification step includes at least one of the following step: anionic-exchange chromatography, hydroxyapatite chromatography or size exclusion chromatography.
It is another object of the invention a process for the production of the recombinant human long pentraxin PTX3protein comprising:

    a) co-transfecting contemporarily or sequentially human cells with a first vector having essentially the sequence of SEQ ID NO: 1and a second vector having essentially the sequence of SEQ ID NO: 2;
    b) selecting and growing the double transfected cells;
    b) purifying the human long pentraxin PTX3protein from the culture medium of double transfected cells.


It is another object of the invention a process for the production the recombinant human long pentraxin PTX3 protein comprising the step of growing the recombinant MS24PTX clone and purifying the human long pentraxin PTX3 protein from the culture medium.



The invention will be now illustrated by means of non limiting examples, referring in particular to the following figures:
FIG. 1: pSASSI-hPTX3 map and main features
FIG. 2: Growth, viability and productivity in Spinner Flask of (A) MS24PTX clone and (B) 293F/PTX3/2F12 clone.
FIG. 3: characterization by SDS-PAGE gradient 4-15% (A) and Size Exclusion Chromatography (B) of recombinant human PTX3 purified from MS24PTX clone.
FIG. 4: FGF2 binding capability of hPTX3 clone 293F/PTX3/2F12 and hPTX3 clone MS24PTX.
FIG. 5: pSC1-hPTX3 map and main features.



EXAMPLES
Example 1
Clone 293F/PTX3/2F12
Construction of the Plasmid pSC1-PTX3
1. Construction of pSG/Ub
1.1 Preparation of the Human Ubiquitin C Promoter Sequence
The human ubiquitin C promoter is taken from pUB/Bsd plasmid (Invtrogen, Cat. n. V512-20), by amplification with PCR. As part of the cloning strategy, recognition sequences for restriction endonucleases are introduced at both ends. A BsaAI site is built in the upstream amplification primer and an EcoRI site in the downstream primer. The amplified region corresponds to nucleotides 1941 to 3161 in the sequence of pUB/Bsd.
The oligonucleotides are designed as follows:









5′p UbC: length: 26mer



(SEQ ID NO: 3)



ATATCACGTG ATC TGG CCT CCG CGC C



 



3′p UbC: length: 23mer



(SEQ ID NO: 4)



GGAATTC GGT CCG GTC TAA CAA A






The protocol for amplification is the following: 1 ng/μl of plasmid DNA, 2 mM MgCl2, 0.2 mM dNTPs, 400 nM of each primer, 1× supplied buffer and 0.04 u/ml of Taq DNA polymerase (Sigma Genosys); temperature profile: 3 min 94° C., 30 times (30 sec. 94° C., 30 sec. 46° C., 2 min 72° C.), 5 min 72° C., cooling at 4° C. until further use.
The amplification product (1238 bp) is purified by silica membrane spin column (NucleoSpin, Machery-Nagel GmbH & Co.), ligated in pGEM-T-Easy vector (Promega Cat. n. A1360) and transformed into E. coli host strain HB2151 (Pharmacia Biotech). Transformants are selected by growth on LB medium supplemented with 50 mg/l ampicillin
Plasmids DNA, isolated from ampicillin resistant colonies, are checked by restriction analysis with StuI plus SacI enzymes (expected  ̃3650 and 600 bp fragments)
Plasmids showing the correct restriction pattern are further checked by sequence analysis of the entire insert and subsequently digested with EcoRI (Sigma-Genosys) and BsaAI (New England Biolabs) restriction enzymes.
Human Ubiquitin C promoter is purified via agarose gel separation and elution on silica membrane spin column.
1.2 Preparation of the Vector Fragment pSG5
Plasmid pSG5 (4076 bp, Stratagene) was cut with the restriction enzymes EcoRI (Sigma-Genosys) and BsaAI (New England Biolabs); the resulting fragments are 1432 and 2644 bp long. The 2644 bp fragment, containing the backbone of pSG5, was prepared and purified via agarose gel electrophoresis plus silica membrane spin column.
1.3 Preparation of pSG/Ub
DNA fragments prepared in steps 1.1 and 1.2 were ligated using T4 DNA ligase (Promega) and transformed in HB2151 E. coli cells. Transformants were selected by growth on LB medium supplemented with 50 mg/l ampicillin.
Plasmid DNA, isolated from ampicillin resistant colonies, was checked by restriction analysis with EcoRI plus SacII enzymes (expected: 2670 and 1192 bp fragments). A plasmid DNA, with the expected restriction pattern, was designed as pSG/Ub.
2. Construction of pSC1
2.1 Preparation of the Neomycin Resistance Cassette (NeoR)
The Neomycin Resistance Cassette (NeoR) was taken from pcDNA3 plasmid (5446 bp, Invitrogen), amplifying it by PCR. As part of the cloning strategy, recognition sequences for restriction endonuclease AflIII were introduced at both ends. The amplified region corresponds to nucleotides 1788 to 3252 in the sequence of pcDNA3 and includes the SV40 promoter and origin of replication, the neomycin resistance ORF, and the SV40 poliA signal.
The oligonucleotides are designed as follows:









5′NeoR



(SEQ ID NO: 5)



ATATACATG TCC CCA GGC AGG CAG AA



 



3′NeoR



(SEQ ID NO: 6)



ATATACAT GTAT ACA GAC ATG ATA AG






Protocol for amplification was the following: 1 ng/μl of plasmid DNA, 2 mM MgCl2, 0.2 mM dNTPs, 400 nM of each primer, 1× supplied buffer and 0.04 u/μl of Taq DNA polymerase (Sigma Genosys); temperature profile: 3 min 94° C., 30 times (30 sec. 94° C., 30 sec. 46° C., 2 min 72° C.), 5 min 72° C., cooling at 4° C. until further use.
The amplification product (1484 bp) was purified by silica membrane spin column, ligated in pGEM-T-Easy vector (Promega Cat. n. A1360) and transformed into E. coli host strain HB2151. Transformants are selected by growth on LB medium, supplemented with 50 mg/l ampicillin Plasmids DNA, isolated from ampicillin resistant colonies, are checked by restriction analysis with SmaI plus SacI enzymes (expected  ̃1200 and 3300 bp fragments).
Plasmids showing the correct restriction pattern were further checked by sequence analysis of the entire insert and subsequently digested with AflIII (New England Biolabs) restriction enzymes. NeoR cassette (1471 bp) was purified via agarose gel separation and elution on silica membrane spin column.
2.2 Preparation of the Vector Fragment pSG/Ub
Plasmid pSG/Ub, prepared in step 1.3, was linearized by AflIII digestion and purified on silica membrane spin column.
2.3 Preparation of pSC1 DNA fragments prepared as in steps 2.1 and 2.2 were ligated using T4 DNA ligase (Promega) and transformed in JM109 E. coli strain (New England Biolabs). Transformants were selected by growth on LB medium, supplemented with 50 mg/l ampicillin.
Antibiotic resistant colonies were preliminarliy analyzed by PCR amplification with 5′NeoR and 3′NeoR oligonucleotides, as previously described, and subsequently, purified plasmids were checked by restriction analysis. For this purpose, SmaI (position 602, inside NeoR sequence) and SacII (position 4142, inside UbC sequence) enzymes were used. A plasmid DNA, with the expected restriction pattern (3540 and 1793 bp fragments), was designed as pSC1.
3. Construction of pSC1-PTX3
3.1 Preparation of the hPTX3 Coding Sequence
The hPTX3(GENBANK® Accession Number BD 131701) sequence was taken from pSG5PTX3(WO 99/32516“Pharmaceutical compositions containing the long pentraxin PTX3) by BamHI (Roche Applied Science) digestion. Human PTX3fragment (1463bp) was purified by agarose gel electrophoresis and silica membrane spin column.
3.2 Preparation of the Vector Fragment pSC1
The pSC1 vector was linearized by BamHI digestion and purified on silica membrane spin column.
3.3 Construction and Verification on pSC1-PTX3
DNA fragments prepared in steps 3.2 and 3.3 were ligated using T4 DNA ligase (Roche Applied Science) and transformed in JM109E. coli strain. Transformants were selected by growth on LB medium, supplemented with 50 mg/l ampicillin and preliminarily screened by PCR with two oligonucleotides complementary to PTX3 sequence.
The oligonucleotides sequences are:









5′PTX



(SEQ ID NO: 7)



GTGAGAACTCGGATGATTATGAT



 



3′PTX



(SEQ ID NO: 8)



TGAAACATACTGAGCTCCTCCAT






In a final volume of 10 μl, reagents for amplification were: 1 μl of boiled colony (1 colony in 50 ml of water), 2 mM MgCl2, 0.2 mM dNTPs, 320 nM of each primer, 0.06% Formamide, 1× supplied buffer and 0.08 u/μl of Taq DNA polymerase (Sigma Genosys); temperature profile: 3 min 96° C., 30 times (30 sec. 94° C., 30 sec. 58° C., 2 min 72° C.), 5 min 72° C., cooling at 4° C. until further use.
Plasmid purified from colonies positive to PCR screening, were digested with SalI restriction enzyme (Roche Applied Science) to check the orientation of hPTX3 insert. A plasmid with the expected restriction pattern (6619 and 177 bp) was sequenced in the regions coding for UbC promoter, NeoR cassette and hPTX3 and identified as pSC1-PTX3.
The new plasmid (pSC1-PTX3) was then constructed with PTX3 cDNA sequence located under ubiquitin promoter control and neomycin resistance gene under SV40 promoter control; all other features and plasmid map are represented in FIG. 1.
The complete sequence of pSC1-PTX3is as follows (SEQ NO: ID 2). The pSC1-hPTX3sequence is represented starting from the first EcoRI site (FIG. 5). The sequence deriving from pSG5containing PTX3cDNA is underlined. The starting codon (ATG) and termination codon are in bold.
pSC1-PTX3 (SEQ ID NO:2)








pSC1-PTX3



(SEQ ID NO: 2)



AATTCGGATCCCCCGGGCTGCAGGAATTCCGGCTCAAACTCAGCTCACTTGAGAGTCTCCTCCCGCCAGCTGTGGAA



 


AGAACTTTGCGTCTCTCCAGCAATGCATCTCCTTGCGATTCTGTTTTGTGCTCTCTGGTCTGCAGTGTTGGCCGAGA


 


ACTCGGATGATTATGATCTCATGTATGTGAATTTGGACAACGAAATAGACAATGGACTCCATCCCACTGAGGACCCC


 


ACGCCGTGCGACTGCGGTCAGGAGCACTCGGAATGGGACAAGCTCTTCATCATGCTGGAGAACTCGCAGATGAGAGA


 


GCGCATGCTGCTGCAAGCCACGGACGACGTCCTGCGGGGCGAGCTGCAGAGGCTGCGGGAGGAGCTGGGCCGGCTCG


 


CGGAAAGCCTGGCGAGGCCGTGCGCGCCGGGGGCTCCCGCAGAGGCCAGGCTGACCAGTGCTCTGGACGAGCTGCTG


 


CAGGCGACCCGCGACGCGGGCCGCAGGCTGGCGCGTATGGAGGGCGCGGAGGCGCAGCGCCCAGAGGAGGCGGGGCG


 


CGCCCTGGCCGCGGTGCTAGAGGAGCTGCGGCAGACGCGAGCCGACCTGCACGCGGTGCAGGGCTGGGCTGCCCGGA


 


GCTGGCTGCCGGCAGGTTGTGAAACAGCTATTTTATTCCCAATGCGTTCCAAGAAGATTTTTGGAAGCGTGCATCCA


 


GTGAGACCAATGAGGCTTGAGTCTTTTAGTGCCTGCATTTGGGTCAAAGCCACAGATGTATTAAACAAAACCATCCT


 


GTTTTCCTATGGCACAAAGAGGAATCCATATGAAATCCAGCTGTATCTCAGCTACCAATCCATAGTGTTTGTGGTGG


 


GTGGAGAGGAGAACAAACTGGTTGCTGAAGCCATGGTTTCCCTGGGAAGGTGGACCCACCTGTGCGGCACCTGGAAT


 


TCAGAGGAAGGGCTCACATCCTTGTGGGTAAATGGTGAACTGGCGGCTACCACTGTTGAGATGGCCACAGGTCACAT


 


TGTTCCTGAGGGAGGAATCCTGCAGATTGGCCAAGAAAAGAATGGCTGCTGTGTGGGTGGTGGCTTTGATGAAACAT


 


TAGCCTTCTCTGGGAGACTCACAGGCTTCAATATCTGGGATAGTGTTCTTAGCAATGAAGAGATAAGAGAGACCGGA


 


GGAGCAGAGTCTTGTCACATCCGGGGGAATATTGTTGGGTGGGGAGTCACAGAGATCCAGCCACATGGAGGAGCTCA


 


GTATGTTTCATAAATGTTGTGAAACTCCACTTGAAGCCAAAGAAAGAAACTCACACTTAAAACACATGCCAGTTGGG


 


AAGGTCTGAAAACTCAGTGCATAATAGGAACACTTGAGACTAATGAAAGAGAGAGTTGAGACCAATCTTTATTTGTA


 


CTGGCCAAATACTGAATAAACAGTTGAAGGAAAGACATTGGAAAAAGCTTATCGATACCGTCGACCTCGAGGGGGGG


 


CCCGGGGATCCAGATCTTATTAAAGCAGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA


 


CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAT


 


GTCTGGTCGACTCTAGACTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT


 


ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTCCCCAGGCA


 


GGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAG


 


AAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC


 


CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCC


 


TCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTAT


 


ATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTT


 


CTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTG


 


TTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGA


 


CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGG


 


GAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTA


 


TCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACA


 


TCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGC


 


TCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGAT


 


GCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGA


 


CCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCG


 


TGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGA


 


CTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTAT


 


GAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTT


 


CTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATA


 


AAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACATGTG


 


AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG


 


ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC


 


CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG


 


AAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG


 


TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC


 


GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT


 


GAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG


 


GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG


 


ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA


 


CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT


 


TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA


 


GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACC


 


ATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG


 


CCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCT


 


AGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC


 


GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAG


 


CGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA


 


CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG


 


AGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT


 


TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG


 


ATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG


 


AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCTTTTTTCAATATT


 


ATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG


 


GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAA


 


TAGGCGTATCACGAGGCCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCG


 


GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGG


 


GTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGC


 


ACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTT


 


GTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATA


 


GGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAAC


 


CGTCTATCAGGGCGATGGCCCACTACGTGATCTGGCCTCCGCGCCGGGTTTTGGCGCCCCCCGCGGGCGCCCCCCTC


 


CTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGC


 


GGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACT


 


CTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGA


 


TCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGG


 


ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGC


 


TTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAG


 


CAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCT


 


TGTGAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTT


 


CGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCAC


 


TGACTGGAGAACTCGGTTTGTCGTCTGTTGCGGGGGCGGCAGTTATGGCGGTGCCGTTGGGCAGTGCACCCGTACCT


 


TTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCG


 


GTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGC


 


GCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGT


 


AGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAA


 


ATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGC


 


TTTTTTGTTAGACCGGACCG






A human cell line (HEK293F) has been chosen for its ability to grow in suspension and in a serum and protein free medium (Florian M Wurm “Production of recombinant protein therapeutics in cultivated mammalian cells” Nature Biotechnology 22(11):1393-1398, 2004, Yan S C et al. “Characterization and novel purification of recombinant human protein C from three mammalian cell lines” Biotechnology (N.Y.) 1990 Jul. 8 (7): 655-61. “Use of cell lines for the production of influenza virus vaccines: an appraisal of technical, manufacturing, and regulatory considerations” Initiative for Vaccine Research, World Health Organization, Geneva, Switzerland (10 Apr. 2007). To transfect HEK293F, pSC1-PTX3 plasmid was used either in a linear (PvuI digested) or in a circular form. The best transfection yield was obtained with linearized plasmid; clones selection was done on a productivity base and growth capability. After several rounds of subcloning the 2F12 clone was selected.
The human clone 2F12, expressing hPTX3, has been deposited at ECACC (European Collection of Cell Cultures, Health Protection Agency, Porton Down, Wiltshire SP4 0JG, UK) on Jan. 10, 2008, pursuant to Budapest Treaty condition under deposit number 08011001. The experimental details are described below.
3.4 Recombinant 293F-Cells Generated from pSC1-PTX3
Transfection and Subcloning
106 cells/ml 293F (Invitrogen cat n° R790-07) were seeded in a 125ml spinner flask in a final FREESTYLETM medium volume of 28ml the day of transfection. The pSC1/PTX3plasmid was then allowed to adsorb to the 293FECTINTM reagent (GIBCO/Invitrogen) according to the manufacturer's protocol.
In brief, in two separate tubes, 30 μg of pSC1-PTX3circular or PvuI linearized were diluted in 1ml of OPTIMEM® (GIBCO/Invitrogen, Carlsbad, CA, USA) and 40 μl of 293FECTINTM (Invitrogen) diluted to 1ml with OPTIMEM® . Both solutions were incubated for 5minutes at room temperature then mixed (final volume 2ml) and incubated for 30minutes in the same conditions. DNA/lipid cocktail was added to cells and incubated at 37° C., 5% CO2with agitation (120rpm). After cultivation for 36hours, the medium was changed into selection medium (200ml FREESTYLETM medium+500 μg/ml of G418) and the transfected cells were plated in ten 96wells plates, 200 μl/well. After 15 days highest producers cell-pools were determined by ELISA and amplified in 24wells, 6wells and T25flask.
Recombinant cell-pools obtained were subcloned with 1 cells per well in 96wells plates, in 50% fresh medium and 50% conditioned medium.
Example 2
Clone MS24PTX
Construction of the plasmid pSASSI-hPTX3
1. Construction of pCEPlightΔ
pCEP4 plasmid (Invitrogen cat. n. V044-50), in which was previously cloned an antibody light chain, loosing a portion of the Multiple Cloning Site and the BamHI restriction site, was cut with the restriction enzymes EcoRV and ClaI (Roche Applied Science); the digestion allowed to obtain a plasmid without the Epstein-Barr-Virus replication origin (oriP) and the nuclear antigen (encoded by the EBNA-1 gene) that permit extrachromosomal replication. The resulting fragments were 6910 and 4281 bp long. The 6910 bp fragment, containing the backbone of pCEP, was purified via agarose gel electrophoresis plus silica membrane spin column. Since ClaI generates sticky end, the fragment was filled in, using T4 DNA polymerase (Roche Applied Science) with the follow protocol: 150 ng of ClaI/EcoRV purified fragment (38 μl), 5 μl of 10× T4 DNA polymerase buffer, 4 μl of dNTP mix 2.5 mM, 3 μl of T4 DNA polymerase (1U/μl). After 15 minutes at 37° C., the reaction was stopped at 70° C. for 5 minutes then on ice. The fragment was purified on a silica membrane spin column and ligated on itself over night at room temperature, by using T4 DNA ligase (Promega). TOP10 competent cells (Invitrogen) were transformed with the ligation mixture and transformants selected by growth on LB plates supplemented with 100 mg/L ampicillin.
Plasmid DNA, isolated from ampicillin resistant colonies, was designed as pCEPlightΔ.
2. Preparation of Vector Fragment Containing Hygromycin Resistance and CMV Promoter.
The Hygromycin Resistance Cassette together with the cytomegalovirus (CMV) immediate early enhancer/promoter was taken from pCEPlightΔ amplifying it by PCR. As a part of the cloning strategy, recognition sequence for restriction endonuclease BamHI was introduced in the oligonucleotide annealing to the 3′ end of CMV promoter.
The amplified region about 5500 bp, included CMV promoter, Hygromycin gene under the control of TK promoter together with TK polyA signal.
The oligonucleotide are designed as follows:









oligo CMV



(SEQ ID NO: 9)



5′GAGAACTGTAACGTTGGATCCAGCTGG 3′



 



oligo H



(SEQ ID NO: 10)



5′GTGTACAAAGGATCCAGACATGATAAG 3′






Protocol for amplification was the following: 2 ng of pCEPlightΔ, 200 nM of each primer, 0.2 mM dNTPs, 1× supplied buffer, 1.5 μl DMSO, 0.5 μl Taq DNA polymerase (Phusion), final volume 50 μl; temperature profile: 1 min 98° C., 35 times (10 sec. 98° C., 30 sec. 55° C., 3 min. 72° C.), 10 min. 72° C., cooling at 4° C. until further use.
The amplification product ( ̃5500 bp) was purified via agarose gel electrophoresis plus silica membrane spin column. Purified fragment was ligated to itself and use to transform TOP10 competent cells (Invitrogen). Plasmid DNA, isolated from ampicillin resistant colonies, was checked by restriction and sequence analysis. and was designed as pCEPΔBam.
3. Preparation of hPTX3 Gene
The hPTX3(GENBANK® Accession Number BD 131701) sequence was taken from pSC1-PTX3as indicated above by BamHI (Roche Applied Science) digestion. Human PTX3fragment (1463bp) was purified by agarose gel electrophoresis and silica membrane spin column.
4. Preparation of pCEPΔBam-hPTX3
The pCEPΔBam vector was linearized by BamHI digestion and purified on silica membrane spin column. pCEPΔBam linearized and DNA fragment corresponding to hPTX3 gene prepared in step 3 were ligated using T4 DNA ligase (Roche Applied Science) and used to transform TOP10 E. coli strain. Transformants were selected by growth on LB medium, supplemented with 100 mg/L ampicillin and preliminarily screened restriction analysis to evaluate PTX3 fragment orientation.
5. Preparation of SV40 Polyadenilation Signal
The SV40 polyA signal was taken from pCEPΔlight plasmid amplifying it by PCR. As part of the cloning strategy, recognition sequences for restriction endonucleoases HindIII and XhoI were introduced at fragment ends respectively.
The oligonucleotides were designed as follows:









PCEPSVH



(SEQ ID NO: 11)



5′AAGCTTAGACATGATAAGATACATTG 3′



 



PCEPSVX



(SEQ ID NO: 12)



5′CTCGAGAGTCGACCGGTCATGGCTGC 3′






Protocol for amplification was the following: 1 ng of pCEPlightΔ, 200 nM of each primer, 0.2 mM dNTPs, 1× supplied buffer, 2 μl MgCl2 50 mM, 0.5 μl Taq DNA polymerase (Invitrogen), final volume 50 μl; temperature profile: 1 min 94° C., 30 times (30 sec. 94° C., 1 min. 55° C., 1 min. 72° C.), 15 min. 72° C., cooling at 4° C. until further use.
The amplification product ( ̃420 bp) was purified via agarose gel electrophoresis plus silica membrane spin column.
6. Preparation of pSASSI-hPTX3 Purified fragment corresponding to SV40 polyA signal and pCEPΔBam-hPTX3 were digested with HindIII/XhoI restriction enzymes and ligated using T4 DNA ligase (Promega). Ligation mixture was used to transform TOP10 competent cells (Invitrogen) and transformants were selected by growth on LB plates containing 100 mg/L of ampicillin.
Plasmid DNA isolated from ampicillin resistant colonies, was checked by restriction and sequence analysis and the plasmid was designated pSASSI-hPTX3.
The complete sequence of pSASSI-HPTX3is as follows (SEQ ID NO: 1). The pSASSI-hPTX 3sequence is represented starting from the BamHI site (FIG. 1). The sequence of hPTX3is in small letters. The starting codon (atg) and the termination codon (taa) are in bold.
pSASSI-HPTX3 (SEQ ID NO: 1)








pSASSI-HPTX3.



(SEQ ID NO: 1)










GGATCCCCCG GGCTGCAGGA ATTCCGGCTC AAACTCAGCT CACTTGAGAG TCTCCTCCCG
  60



 


CCAGCTGTGG AAAGAACTTT GCGTCTCTCC AGCAATGCAT CTCCTTGCGA TTCTGTTTTG
 120


 


TGCTCTCTGG TCTGCAGTGT TGGCCGAGAA CTCGGATGAT TATGATCTCA TGTATGTGAA
 180


 


TTTGGACAAC GAAATAGACA ATGGACTCCA TCCCACTGAG GACCCCACGC CGTGCGACTG
 240


 


CGGTCAGGAG CACTCGGAAT GGGACAAGCT CTTCATCATG CTGGAGAACT CGCAGATGAG
 300


 


AGAGCGCATG CTGCTGCAAG CCACGGACGA CGTCCTGCGG GGCGAGCTGC AGAGGCTGCG
 360


 


GGAGGAGCTG GGCCGGCTCG CGGAAAGCCT GGCGAGGCCG TGCGCGCCGG GGGCTCCCGC
 420


 


AGAGGCCAGG CTGACCAGTG CTCTGGACGA GCTGCTGCAG GCGACCCGCG ACGCGGGCCG
 480


 


CAGGCTGGCG CGTATGGAGG GCGCGGAGGC GCAGCGCCCA GAGGAGGCGG GGCGCGCCCT
 540


 


GGCCGCGGTG CTAGAGGAGC TGCGGCAGAC GCGAGCCGAC CTGCACGCGG TGCAGGGCTG
 600


 


GGCTGCCCGG AGCTGGCTGC CGGCAGGTTG TGAAACAGCT ATTTTATTCC CAATGCGTTC
 660


 


CAAGAAGATT TTTGGAAGCG TGCATCCAGT GAGACCAATG AGGCTTGAGT CTTTTAGTGC
 720


 


CTGCATTTGG GTCAAAGCCA CAGATGTATT AAACAAAACC ATCCTGTTTT CCTATGGCAC
 780


 


AAAGAGGAAT CCATATGAAA TCCAGCTGTA TCTCAGCTAC CAATCCATAG TGTTTGTGGT
 840


 


GGGTGGAGAG GAGAACAAAC TGGTTGCTGA AGCCATGGTT TCCCTGGGAA GGTGGACCCA
 900


 


CCTGTGCGGC ACCTGGAATT CAGAGGAAGG GCTCACATCC TTGTGGGTAA ATGGTGAACT
 960


 


GGCGGCTACC ACTGTTGAGA TGGCCACAGG TCACATTGTT CCTGAGGGAG GAATCCTGCA
1020


 


GATTGGCCAA GAAAAGAATG GCTGCTGTGT GGGTGGTGGC TTTGATGAAA CATTAGCCTT
1080


 


CTCTGGGAGA CTCACAGGCT TCAATATCTG GGATAGTGTT CTTAGCAATG AAGAGATAAG
1140


 


AGAGACCGGA GGAGCAGAGT CTTGTCACAT CCGGGGGAAT ATTGTTGGGT GGGGAGTCAC
1200


 


AGAGATCCAG CCACATGGAG GAGCTCAGTA TGTTTCATAA ATGTTGTGAA ACTCCACTTG
1260


 


AAGCCAAAGA AAGAAACTCA CACTTAAAAC ACATGCCAGT TGGGAAGGTC TGAAAACTCA
1320


 


GTGCATAATA GGAACACTTG AGACTAATGA AAGAGAGAGT TGAGACCAAT CTTTATTTGT
1380


 


ACTGGCCAAA TACTGAATAA ACAGTTGAAG GAAAGACATT GGAAAAAGCT TAGACATGAT
1440


 


AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA AATGCTTTAT
1500


 


TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA ATAAACAAGT
1560


 


TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGGTGT GGGAGGTTTT
1620


 


TTAAAGCAAG TAAAACCTCT ACAAATGTGG TATGGCTGAT TATGATCCGG CTGCCTCGCG
1680


 


CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC GGTCACAGCT
1740


 


TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC
1800


 


GGGTGTCGGG GCGCAGCCAT GACCGGTCGA CTCTCGAGGG GGGGCCCGGG GATCCAACGT
1860


 


TACAGTTCTC CAGTGCATGT AATCCCTTCA GTTGGTTGGT ACAACTTGCC AACTGGGCCC
1920


 


TGTTCCACAT GTGACACGGG GGGGGACCAA ACACAAAGGG GTTCTCTGAC TGTAGTTGAC
1980


 


ATCCTTATAA ATGGATGTGC ACATTTGCCA ACACTGAGTG GCTTTCATCC TGGAGCAGAC
2040


 


TTTGCAGTCT GTGGACTGCA ACACAACATT GCCTTTATGT GTAACTCTTG GCTGAAGCTC
2100


 


TTACACCAAT GCTGGGGGAC ATGTACCTCC CAGGGGCCCA GGAAGACTAC GGGAGGCTAC
2160


 


ACCAACGTCA ATCAGAGGGG CCTGTGTAGC TACCGATAAG CGGACCCTCA AGAGGGCATT
2220


 


AGCAATAGTG TTTATAAGGC CCCCTTGTTA ACCCTAAACG GGTAGCATAT GCTTCCCGGG
2280


 


TAGTAGTATA TACTATCCAG ACTAACCCTA ATTCAATAGC ATATGTTACC CAACGGGAAG
2340


 


CATATGCTAT CGAATTAGGG TTAGTAAAAG GGTCCTAAGG AACAGCGATC GATGATAAGC
2400


 


TGTCAAACAT GAGAATTCTT GAAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT
2460


 


TAATGTCATG ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG
2520


 


CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA
2580


 


ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT
2640


 


CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA
2700


 


AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA
2760


 


ACTGGATCTC AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT
2820


 


GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGTTG ACGCCGGGCA
2880


 


AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT
2940


 


CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC
3000


 


CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT
3060


 


AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA
3120


 


GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGCAG CAATGGCAAC
3180


 


AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT
3240


 


AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG
3300


 


CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC
3360


 


ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC
3420


 


AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG
3480


 


GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA
3540


 


ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG
3600


 


TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA
3660


 


TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT
3720


 


GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG
3780


 


AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA
3840


 


CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG
3900


 


TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA
3960


 


GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC
4020


 


CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA
4080


 


GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC
4140


 


AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG
4200


 


TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC
4260


 


CTTTTTACGG TTCCTGGCCT TTTGCTGCGC CGCGTGCGGC TGCTGGAGAT GGCGGACGCG
4320


 


ATGGATATGT TCTGCCAAGG GTTGGTTTGC GCATTCACAG TTCTCCGCAA GAATTGATTG
4380


 


GCTCCAATTC TTGGAGTGGT GAATCCGTTA GCGAGGTGCC GCCGGCTTCC ATTCAGGTCG
4440


 


AGGTGGCCCG GCTCCATGCA CCGCGACGCA ACGCGGGGAG GCAGACAAGG TATAGGGCGG
4500


 


CGCCTACAAT CCATGCCAAC CCGTTCCATG TGCTCGCCGA GGCGGCATAA ATCGCCGTGA
4560


 


CGATCAGCGG TCCAGTGATC GAAGTTAGGC TGGTAAGAGC CGCGAGCGAT CCTTGAAGCT
4620


 


GTCCCTGATG GTCGTCATCT ACCTGCCTGG ACAGCATGGC CTGCAACGCG GGCATCCCGA
4680


 


TGCCGCCGGA AGCGAGAAGA ATCATAATGG GGAAGGCCAT CCAGCCTCGC GTCGCGAACG
4740


 


GCGAACGCCA GCAAGACGTA GCCCAGCGCG TCGGCCGCCA TGCCCTGCTT CATCCCCGTG
4800


 


GCCCGTTGCT CGCGTTTGCT GGCGGTGTCC CCGGAAGAAA TATATTTGCA TGTCTTTAGT
4860


 


TCTATGATGA CACAAACCCC GCCCAGCGTC TTGTCATTGG CGAATTCGAA CACGCAGATG
4920


 


CAGTCGGGGC GGCGCGGTCC CAGGTCCACT TCGCATATTA AGGTGACGCG TGTGGCCTCG
4980


 


AACACCGAGC GACCCTGCAG CGACCCGCTT AACAGCGTCA ACAGCGTGCC GCAGATCCCG
5040


 


GGCAATGAGA TATGAAAAAG CCTGAACTCA CCGCGACGTC TGTCGAGAAG TTTCTGATCG
5100


 


AAAAGTTCGA CAGCGTCTCC GACCTGATGC AGCTCTCGGA GGGCGAAGAA TCTCGTGCTT
5160


 


TCAGCTTCGA TGTAGGAGGG CGTGGATATG TCCTGCGGGT AAATAGCTGC GCCGATGGTT
5220


 


TCTACAAAGA TCGTTATGTT TATCGGCACT TTGCATCGGC CGCGCTCCCG ATTCCGGAAG
5280


 


TGCTTGACAT TGGGGAATTC AGCGAGAGCC TGACCTATTG CATCTCCCGC CGTGCACAGG
5340


 


GTGTCACGTT GCAAGACCTG CCTGAAACCG AACTGCCCGC TGTTCTGCAG CCGGTCGCGG
5400


 


AGGCCATGGA TGCGATCGCT GCGGCCGATC TTAGCCAGAC GAGCGGGTTC GGCCCATTCG
5460


 


GACCGCAAGG AATCGGTCAA TACACTACAT GGCGTGATTT CATATGCGCG ATTGCTGATC
5520


 


CCCATGTGTA TCACTGGCAA ACTGTGATGG ACGACACCGT CAGTGCGTCC GTCGCGCAGG
5580


 


CTCTCGATGA GCTGATGCTT TGGGCCGAGG ACTGCCCCGA AGTCCGGCAC CTCGTGCACG
5640


 


CGGATTTCGG CTCCAACAAT GTCCTGACGG ACAATGGCCG CATAACAGCG GTCATTGACT
5700


 


GGAGCGAGGC GATGTTCGGG GATTCCCAAT ACGAGGTCGC CAACATCTTC TTCTGGAGGC
5760


 


CGTGGTTGGC TTGTATGGAG CAGCAGACGC GCTACTTCGA GCGGAGGCAT CCGGAGCTTG
5820


 


CAGGATCGCC GCGGCTCCGG GCGTATATGC TCCGCATTGG TCTTGACCAA CTCTATCAGA
5880


 


GCTTGGTTGA CGGCAATTTC GATGATGCAG CTTGGGCGCA GGGTCGATGC GACGCAATCG
5940


 


TCCGATCCGG AGCCGGGACT GTCGGGCGTA CACAAATCGC CCGCAGAAGC GCGGCCGTCT
6000


 


GGACCGATGG CTGTGTAGAA GTACTCGCCG ATAGTGGAAA CCGACGCCCC AGCACTCGTC
6060


 


CGAGGGCAAA GGAATAGGGG AGATGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC
6120


 


CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA TAAAACGCAC GGGTGTTGGG
6180


 


TCGTTTGTTC ATAAACGCGG GGTTCGGTCC CAGGGCTGGC ACTCTGTCGA TACCCCACCG
6240


 


AGACCCCATT GGGGCCAATA CGCCCGCGTT TCTTCCTTTT CCCCACCCCA CCCCCCAAGT
6300


 


TCGGGTGAAG GCCCAGGGCT CGCAGCCAAC GTCGGGGCGG CAGGCCCTGC CATAGCCACT
6360


 


GGCCCCGTGG GTTAGGGACG GGGTCCCCCA TGGGGAATGG TTTATGGTTC GTGGGGGTTA
6420


 


TTATTTTGGG CGTTGCGTGG GGTCTGGTCC ACGACTGGAC TGAGCAGACA GACCCATGGT
6480


 


TTTTGGATGG CCTGGGCATG GACCGCATGT ACTGGCGCGA CACGAACACC GGGCGTCTGT
6540


 


GGCTGCCAAA CACCCCCGAC CCCCAAAAAC CACCGCGCGG ATTTCTGGCG TGCCAAGCTA
6600


 


GTCGACCAAT TCTCATGTTT GACAGCTTAT CATCGCAGAT CCGGGCAACG TTGTTGCCAT
6660


 


TGCTGCAGGC GCAGAACTGG TAGGTATGGA AGATCTATAC ATTGAATCAA TATTGGCAAT
6720


 


TAGCCATATT AGTCATTGGT TATATAGCAT AAATCAATAT TGGCTATTGG CCATTGCATA
6780


 


CGTTGTATCT ATATCATAAT ATGTACATTT ATATTGGCTC ATGTCCAATA TGACCGCCAT
6840


 


GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA
6900


 


GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC
6960


 


CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG
7020


 


GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC
7080


 


ATCAAGTGTA TCATATGCCA AGTCCGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG
7140


 


CCTGGCATTA TGCCCAGTAC ATGACCTTAC GGGACTTTCC TACTTGGCAG TACATCTACG
7200


 


TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA GTACACCAAT GGGCGTGGAT
7260


 


AGCGGTTTGA CTCACGGGGA TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT
7320


 


TTTGGCACCA AAATCAACGG GACTTTCCAA AATGTCGTAA TAACCCCGCC CCGTTGACGC
7380


 


AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTCGT TTAGTGAACC
7440


 


GTCAGATCTC TAGAAGCTGG GTACCAGCT
7469






Example 3
Recombinant Ms24PTX Clone Generated by pSASSI-hPTX3 Transfection
1. Transfection and Subcloning
106 cells/ml 293F/PTX3/2F12were seeded in a 125ml spinner flask in a final FREESTYLETM medium volume of 28 ml the day of transfection. The pSASSI-hPTX3 plasmid was then allowed to adsorb to the 293FECTINTM reagent (GIBCO/Invitrogen) according to the manufacturer's protocol.
In brief, 30 μg of pSASSI-hPTX3NruI linearized was diluted in 1ml of OPTIMEN ® (GIBCO/Invitrogen, Carlsbad, CA, USA) and 40 μl of 293FECTINTM (Invitrogen) diluted to 1ml with OPTIMEN ®. Both solutions were incubated for 5minutes at room temperature then mixed (final volume 2ml) and incubated for 30 minutes in the same conditions. DNA/lipid cocktail was added to cells and incubated at 37° C., 5% CO2with agitation 120rpm). After cultivation for 36hours, the medium was changed into selection medium (200ml FREESTYLETM medium+200 μg/ml of Hygromycin) and the transfected cells were plated in ten 96wells plates, 200 μl/well. After 15days highest producers cell-pools were determined by ELISA and amplified in 24wells, 6wells and T25flask.
Recombinant cell-pools obtained were subcloned with 1 cells per well in 96wells plates, in 50% fresh medium and 50% conditioned medium.
2. ELISA Detection of Recombinant hPTX3
Purified PTX3or PTX3secreted in the culture supernatant were titrated using a sandwich ELISA. To detect PTX3, 96-well Nunc MAXISORB® microtiter plates (Nunc, Roskilde, Denmark) were coated overnight, at 4° C., with 700ng/ml of the rat monoclonal antibody MNB4anti-human PTX3(AlexisTM Biochemicals, Lausen, Switzerland) in 15 mM sodium carbonate buffer, pH 9.6. Wells were washed with PBS plus 0.05% TWEEN®-20 (PBS-Tw, washing solution) and blocked with 300 μl of PBS-Tw containing 5% dry milk, for 2hours at room temperature.
Cell supernatants or purified recombinant human PTX3 were added to the wells, diluted in washing solution plus 1% BSA. A standard curve, made with purified recombinant human PTX3 from CHO cells, ranging from 0 to 100 ng/ml, was done for quantification. After 1 hour of incubation at 37° C., bound PTX3 was detected using biotin-conjugated polyclonal rabbit anti-PTX 3 antibody, followed by incubation with streptavidin conjugated to horseradish peroxidase (Sigma-Aldrich, USA). Finally 2.2′-azino-bis 3-ethylbenxthiazoline-6-sulfonic acid (Sigma Chemical Co. USA) was added for color development and optical density at 405 nm was assessed using a Microplate Reader Model 3550 EIA (Bio-Rad, Hercules, Calif., USA).
Example 4
Comparison of Growth, Viability and Productivity of Clones 293F/PTX3/2F12 and MS24PTX
Cells deriving from the two clones were seeded at a density of 1.000.000cells/ml (viability ≧90%) in a 500ml of FREESTYLETM 293medium in 1liter Spinner flasks. The growing, the viability and the productivity were monitored for about 1week until cells start die.
FIG. 2 shows the growth, viability and productivity of the MS24PTX (panel A) and 293F/PTX3/2F12 (panel B) clones in the same seeding and growing conditions. As shown in the figure, with the re-transfection of the PTX3 expressing clone 293F/PTX3/2F12 with a new plasmid in which PTX3 is under the control of CMV promoter (MS24PTX clone) we were able to obtain about 4 fold increase in PTX3 productivity.
Example 5
Purification of Recombinant Human PTX3 from MS24PTX Clone
Culture supernantant from MS24PTX clone, grown in spinner flask, was loaded onto a Q-SepharoseTM Fast Flow (GE Healthcare, UK) packed column. Retained material was eluted using a nonlinear gradient. The PTX3-containing fraction was directly applied to a ceramic Hydroxyapatite (BioRad, Hercules, Calif., USA) packed column. The retained material was eluted by increasing phosphate concentration in a nonlinear fashion. The PTX3-containing fraction was concentrated and buffer changed on a ultrafiltration membrane (Pellicon-Biomax 100, Millipore) than characterized by Size Exclusion Chromatography on Biosep SEC 54000 (Phenomenex) and SDS-PAGE (FIG. 3).
Example 6
Binding of h-PTX3 to FGF2
The binding of purified recombinant hPTX3 to FGF2 was assessed in an ELISA system. A 96-wells plate (Falcon 3912) was coated with 2 μg/ml of FGF2 (Calbiochem) in PBS and incubated overnight at 4° C. Wells were washed with PBS plus 0.1% Triton X-100 (PBS-Tr, washing solution) and blocked with 200 μl of PBS-Tr containing 3% BSA (PBS-B blocking and diluent solution) for 2 hours at room temperature. After washing, binding was performed adding 100 μl of samples, diluted in PBS-B at PTX3 concentrations ranging from 0 to 120 ng/ml, and incubating the plate at 37° C. for 1 hr. After wash, plates were incubated with 100 μl/well of 100 ng/ml rabbit anti-PTX3 polyclonal antibody (1 hr at 37° C.), washed again and incubated with 100 μl of horseradish peroxidase-labeled goat anti-rabbit IgG (1:1000 in PBS-B; 1 hr at 37° C.). After washing, 100 μl of chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (sigma-Aldrich) were added and after 10-15 min, the reaction was stopped adding 100 μl of HCl 1M and absorbance determined using a Microplate Reader Model 3550 EIA (Bio-Rad, Hercules, Calif., USA). (FIG. 4).
DEPOSIT STATEMENT
Cultures of the following biological material(s) have been deposited with the European Collection of Cell Cultures (ECACC), located at:
Culture Collections
Public Health
England
Porton Down
Salisbury
SP4OJG, UK under conditions that satisfy the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.










International Depository Accession Numbers












ECACC 
Date of 



Deposited Vector
Accession No.
Deposit Accepted







293F/PTX3/2F12
08011001
10 Jan. 2008



MS24PTX
09072902
29 Jul. 2009

## Claims

1. An eukaryotic expression vector comprising the sequence of SEQ ID NO: 1.

2. A recombinant cell able to express human long pentraxin PTX3 protein, wherein said recombinant cell comprises the eukaryotic expression vector of claim 1.

3. The recombinant cell of claim 2, wherein the recombinant cell is a recombinant HEK293F cell line.

4. The recombinant cell of claim 3, wherein the recombinant HEK293F cell line is 293F/PTX3/2F12 clone deposited with European Collection of Cell Cultures (ECACC) as Accession No. 08011001.

5. A recombinant cell able to express human long pentraxin PTX3 protein, wherein said recombinant cell is MS24PTX clone deposited with ECACC as Accession Number 09072902.

6. A method of making a recombinant human host cell able to express human long pentraxin PTX3 protein, comprising transfecting a recombinant human host cell already transfected with a vector capable of expressing human long pentraxin PTX3 protein with the eukaryotic expression vector of claim 1 to produce a double-transfected recombinant human host cell.

7. The method of claim 6, wherein the recombinant human host cell already transfected with a vector capable of expressing human long pentraxin PTX3 is 293F/PTX3/2F12 clone deposited with ECACC as Accession No. 08011001.

8. The method according to claim 6 wherein the eukaryotic expression vector comprising the sequence of SEQ ID NO: 1 is linearized.

9. The method of claim 6, further comprising selecting and growing the double-transfected host cell in a culture medium.

10. A process for producing recombinant human long pentraxin PTX3 protein comprising:
a) co-transfecting contemporarily or sequentially human cells with a first vector having the sequence of SEQ ID NO: 1 and a second vector having the sequence of SEQ ID NO: 2;
b) selecting and growing the double transfected cells in a culture medium; and
c) purifying the human long pentraxin PTX3 protein from the culture medium of the double transfected cells.

11. A method of producing human long pentraxin PTX3 protein, comprising
a) culturing the recombinant host cell of claim 2 in a culture medium; and
b) purifying human long pentraxin PTX3 protein from the culture medium.

12. A method for producing human long pentraxin PTX3 protein, comprising
a) transfecting a recombinant human cell already transfected with an expression vector encoding recombinant human long pentraxin PTX3 protein with an eukaryotic expression vector comprising the sequence of SEQ ID NO: 1;
b) selecting and growing the transfected recombinant human cell comprising the eukaryotic expression vector comprising the sequence of SEQ ID NO: 1 in a culture medium; and
c) purifying human long pentraxin PTX3 protein from the culture medium of step b).

13. The method of claim 12, wherein the recombinant human cell already transfected with an expression vector encoding recombinant human long pentraxin PTX3 protein is a recombinant HEK293F cell line.

14. The method of claim 13, wherein the recombinant HEK293F cell line is 293F/PTX3/2F12 clone deposited with ECACC as Accession No. 08011001.

15. The method of claim 12, wherein said purifying is by at least one of anionic-exchange chromatography, hydroxyapatite chromatography or size exclusion chromatography.

16. A process for producing recombinant human long pentraxin PTX3 protein comprising the steps of growing a recombinant MS24PTX clone deposited with ECACC as Accession No. 09072902 in a culture medium and purifying the human long pentraxin PTX3 ; protein from the culture medium.