GB2094314A

GB2094314A - Recombinant DNA

Info

Publication number: GB2094314A
Application number: GB8203262A
Authority: GB
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1981-02-05
Filing date: 1982-02-04
Publication date: 1982-09-15

Abstract

Recombinant DNA coding for the K99 antigen of enteropathogenic strains of Escherichia coli. Such recombinant DNA could be used to produce sufficient quantities of K99 antigen for incorporation in a vaccine against scouring, which is a diarrhoeal disease of adult and particularly neonatal animals. DNA coding for the E. coli K99 antigen can be obtained from the K99 plasmid of E. coli K12. The DNA can then be cloned in a high copy number plasmid such as pBR322 in E. coli C host cells and expressed to give the desired antigen.

Description

SPECIFICATION Recombinant DNA The present invention relates to recombinant deoxyribonucleic acid (DNA) and, in particular, to recombinant DNA coding for an antigen of the bacterium Escherichia coli.

Scouring is a diarrhoeal disease in neonatal animals often caused by toxins released from enteropathogenic strains of E. colt, which can colonise the gut before the natural flora is established.

Even adult animals, however, are susceptible to this form of diarrhoea when the natural balance of flora in the gut is upset, for example following antibiotic treatment or a sudden change in diet.

An important early stage in the pathogenesis of enterotoxigenic E. coli is the adhesion of the bacterium to the epithelium of the intestinal tract of the animal. Adhesion is mediated by specific capsular antigens present on the surface of the bacterial cell. Examples of such antigens, which have a polypeptide structure, include those designated as K88 and K99 antigens. The K88 antigen is primarily responsible for the adhesion of enteropathogenic strains of E. coli to the intestinal villi of piglets while the K99 antigen is primarily responsible for adhesion of enteropathogenic strains of E. coli to the intestinal villi of calves and lambs, and, to a lesser extent, of piglets. There are also substantial morphological differences between the K88 and K99 antigens.The K88 antigen is a fibrous capsule surrounding the cell, but the K99 antigen is a filamentous pilus. K99 and the various serotype of K88 also have important differing serological properties.

Using the techniques developed over the last five years it is now possible to introduce the DNA coding for specific proteins into bacterial cells via the intermediary of a plasmid or other cloning vehicle; see for example Burrell, C. J., et al, Nature, 279, 43-47, 1979. In general the construction of the recombinant DNA molecules comprises the steps of excising the DNA template coding for the desired protein from a parent cell and inserting this piece of heterologous DNA into a cloning vehicle, such as a bacterial plasmid, and then transforming an appropriate bacterial host with the modified plasmid. A general discussion of the manipulation of genes leading to the formation of recombinant DNA by S. Cohen was published in Scientific American, 233, 24-33, 1975.

Using the above techniques, study of the expression of the genetic determinant that encodes for one particular type of K88 antigen, namely K88ab, has been made by Mooi et al (Nucleic Acids Research 6(3) 1979 (849)).

We have now discovered that genetic manipulation techniques may be applied to produce pilusforming antigens of enteropathogenic strains of E. call. The present invention is particularly but not exclusively concerned with the production of the K99 antigen which, for the reasons outlined above, forms an important immunological component of any vaccine for the treatment or prevention of E. coliinduced diarrhoeal disorders in calves, lambs and to some extent piglets.

Filamentous antigens of especially the K99 antigen of E. coli are generally associated with large, low copy number using conjugative plasmids. However, by using genetic manipulation techniques we have been able to incorporate the DNA nucleotide sequence coding for such antigens into smaller high copy number plasmids whereby increased levels of the antigen may be expressed upon replication.

For a better understanding of the present invention the following glossary is provided for the meaning of certain terms as used in the context of the present invention: Recombinant DNA: a hybrid double stranded DNA molecule comprising at least two double stranded DNA nucleotide sequences, one of the double stranded DNA nucleotide sequences not being found together in nature with the other, or at least one of the other, DNA nucleotide sequences.

Cloning vehicle: non-chromosomal double stranded DNA capable of replicating when placed in a unicellular microorganism.

Expression: the process involved in producing a polypeptide from a gene, e.g. a structural gene.

Plasmid: a cloning vehicle derived from viruses or bacteria.

Expression control sequence: a sequence of DNA nucleotides that controls or regulates the expression of a gene, e.g. a structural gene.

Structural gene: a sequence of DNA nucleotides which codes for a sequence of amino acids characteristic of a specific polypeptide.

According to a first aspect of the present invention, there is provided a recombinant DNA molecule comprising a nucleotide sequence substantially coding for a filamentous pilus antigen of an enteropathogenic strain of Escherichia coli.

The filamentous pilus antigen is desirably K99, and, therefore, there is further provided a recombinant DNA molecule comprising a nucleotide sequence substantially coding for the K99 antigen of an enteropathogenic strain of E. coli. Such a DNA molecule is capable of being expressed as a polypeptide recognisable as substantially corresponding to the K99 antigen.

The recombinant DNA molecule may also contain an expression control sequence or sequences preferably positioned adjacent to the nucleotide sequence coding for the pilus antigen. The control sequence(s) may be derived from E. coli DNA or from a heterologous source.

In the recombinant DNA molecules defined above, the nucleotide sequence substantially coding for the said filamentous pilus antigen is preferably present in a cloning vehicle, conveniently a plasmid having a relatively high copy number and/or a relatively low molecular weight compared with the parent plasmid from which the said sequence is derived. The sequence may be enzymatically excised or chemically synthesised. A preferred cloning vehicle is the plasmid pBR322.

According to a third aspect of the present invention, there is provided a host cell containing a recombinant DNA molecule as defined above.

According to a forth aspect of the present invention, there is provided a method of preparing recombinant DNA molecules as defined above, which comprises inserting a nucleotide sequence substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coli, into a cloning vehicle.

In the above-described method the nucleotide sequence for the filamentous pilus antigen is preferably obtained by digestion with a restriction enzyme of a plasmid containing the desired sequence, e.g. a high molecular weight low copy number plasmid such as p TM 1 obtained for example from E. coiiKl 2. It has been found that Hind Ill restriction enzyme provides a fragment, of about 13.1 Kb (thousands of base pairs), which comprises a nucleotide sequence substantially coding for the desired antigen. Hind Ill cleaves the DNA of the original plasmid at sites where complementary nucleotides are arranged in rotational symmetry, the resuming fragments having what are known in the art as "cohesive" or "sticky" ends.The above-described fragments obtained by restriction enzyme digestion and containing the desired sequence of nucleotides substantially coding for a filamentous pilus antigen represent a further feature of the present invention.

The above-mentioned fragments comprising the desired nucleotide sequence can then be inserted in the cloning vehicle, e.g. a low molecular weight high copy number plasmid such as pBR322 which has previously been treated with a restriction enzyme to provide open ends between which the fragment containing the desired nucleotide sequence may be inserted and ligated, e.g. using a DNA ligase enzyme such as T4 ligase. Opening of the cloning vehicle is preferably effected using Hind Ill restriction enzyme and subsequent treatment with a phosphatase enzyme to remove terminal phosphate groups and thus prevent re-ligation. Other plasmids and indeed bacteriophages may be used as cloning vehicles, but pBR322 is preformed because of its ready availability and the relative ease with which it can be manipulated.

It will be seen from the Example given below that by using the genetic manipulation techniques outlined above, it is possible to obtain 5 different hybrid plasmid forms of the recombinant DNA according to the present invention. These 5 hybrid plasmids are identified by the codes pTP 13, pTP23, pTP33, pTP43 and pTP53 and may be differentiated, as discussed below, on the basis of their differing behaviour on electrophoresis and enzyme digestion. Of the 5 hybrid plasmids, pTP 1 3 and pTP43 are particularly preferred on account of their high copy number in comparison with pTM1 the naturally occuring K99 plasmid, and also their high level of K99 antigen expression in a host cell.The restriction enzyme maps for these two hybrid plasmids as shown in Figure 3 of the accompanying drawings which are discussed more fully below. These two hybrid plasmids, substantially as herein described, thus represent a preferred embodiment of the recombinant DNA according to the present invention.

After synthesis of the recombinant DNA molecules as defined above, an appropriate host, e.g. a bacterium such as E. coli, e.g. E. coli C, is transformed with the recombinant DNA which is then replicated in the host whereby the said nucleotide sequence for the filamentous pilus antigen is expressed as that antigen. A suitable strain of E. coli C is available from Dr. Barbara Bachman, Department of Microbiology School of Medicine, Yale University, New Haven, Connecticut, 06510, U.S.A.

The filamentous pilus antigen expressed by the recombinant DNA according to the present invention may be incorporated in a suitable vaccine for the prevention of E. coli-induced diarrhoeal disorders in lambs, calves and piglets. The formulation of such a vaccine may be carried out using techniques which are conventional in the vaccine art.

For a better understanding of the present invention, and to show how it may be put into effect, the following Example will now be given. Before the results of the Example are given, however, an explanation of the materials and methods used is given.

Example Bacterial strains A K99 positive exconjugant of E. coli Ski 2 was used as a source of K99 plasm id. This plasmid was designated pTM 1. The strain used for cloning the hybrid plasmids was E. coli C, it should be noted that E. coli B41 could also be used.

Culture conditions In order to compare E. coli C strains containing different K99 hybrid plasmids, strains were grown in 20 ml of Trypticase Soy Broth (BBL Microbiology Systems, Cockeysville, Maryland, U.S.A.) in 100 ml baffled conical flasks which were shaken for 18-20 hr. at 140 cycles per min and 370C. Strains were maintained on sheep blood/nutrient agar plates to which antibiotics had been added where appropriate.

Isolation of plasmid DNA Plasmids were routinely prepared by growing bacteria in L-broth (tryptone, 1%; yeast extract, 0.5%; NaCI, 0.5%) overnight at 370C. When pBR322 and hybrid derivatives of pBR322 were prepared, chloramphenicol (200 ug/ml) was added to the bacterial cultures when they had reached an optical density (OD)650 of 0.1 and then incubated for a further 1 8 hr. The bacteria were then centrifuged (7 700 g, 5 min), resuspended in 3.0 ml of 25% (w/v) sucrose in 0.05 M Tris buffer (pH 8.0) and 0.5 ml of lysozyme (5 mg/ml in 0.25 M Tris buffer, pH 8.0) and 1.0 ml of ethylenediaminetetraacetic acid (EDTA) (0.25 M, pH 8.0) were added.After 5 min, 4.0 ml of Brij 58 lysis mixture was added (Brij 58, 1%; sodium deoxycholate, 0.4%; EDTA, 0.0625 M; Tris buffer, 0.05 M (pH 8.0)). After 3-10 incubation at 40C, the lysed bacteria were centrifuged (48 000 g, 1 5 min).

Equilibrium centrifugation in CsCI/ethidium bromide was then carried out by mixing 7.0 ml of the supernatant with 7 g of CsCI and 0.875 ml of ethidium bromide (5 mg/ml). The mixture was transferred to a 10 ml centrifuge tube, overlayed with mineral oil and centrifuged at 40 000 rpm for at least 40 hr a 1 90C. After centrifugation, plasmid bands were located by means of a UV (366 nm) light and removed with a syringe and needle. Ethidium bromide was subsequently removed from the plasmid preparations by two extractions with iso-propanol saturated with CsCI and the latter was then removed by dialysis against several changes of Tris buffer (10 mM, pH 7.6).

Enzymes and chemicals Restriction enzymes, T4 DNA ligase and bacterial alkaline phosphatase as well as agarose were purchased from Bethesda Research Laboratories. ATP, dithiothreitol, ethidium bromide, Tris base, Brij 58, lysozyme and RNase were purchased from Sigma.

Cleavage of DNA and cloning procedures For characterisation, plasmid DNA was cleaved by restriction enzymes in conventional manner.

For the purpose of cloning, DNA was digested for 1-2 hr at 370C in 66.0 mM Tris buffer pH 7.7 which was made 10.0 mM with respect to MgCl2. The vector plasm id, pBR322 was treated with alkaline phosphatase at the same time as restriction enzyme digestion to remove terminal phosphate groups.

Enzymes were then inactivated by incubation at 650C for 10 min. Having checked that the vector was unable to re-ligate to the open circle form, cloning of the restriction enzyme fragments of the K99 plasmid from E. coli K 12 was carried out by adding one fifth the quantity of pBR322 digested with the same restriction enzyme as well as alkaline phosphatase, followed by ATP to a final level of 0.4 mM, dithiothreitol to a final level of 10.0 mM and 1-2 ul of T4 DNA ligase. After overnight incubation at room temperature (200 C), the DNA ligase was inactived by incubation at 650C for 10 min.

Transformation procedure For transformation, a 25.0 ml shake flask culture of E. coli C was grown in nutrient broth at 370C and 140 cycles/min until the OD590 was 0.5-0.6. At this stage, the culture was chilled in ice water, centrifuged (8 000 rpm for 5 min at 40C), washed with 25.0 ml of chilled CaC12 (0.03 M) and resuspended in 25.0 ml of chilled Cacti2. After 20 min incubation on ice, the cells were centrifuged and resuspended in 2.0-5.0 ml of chilled CaCI2. The 20-50 ul of DNA (containing 1-1 000 ng) was added to 0.2 ml of the chilled cell suspension followed by 0.1 ml of CaCI2. After 30 min incubation on ice the bacteria were 'heat pulsed' by 3 min incubation at 420C with agitation.Then 3.0 ml of L-broth (made 0.2% with respect to glucose) was added and the cells were incubated for 90 min at 370C before being plated on appropriate selective media at dilutions ranging from 010-2. Cell controls to which DNA had not been added were routinely run at the same time to ensure that the recipient strain of E. coli remained sensitive to the selective media in the absence of transformation.

Agarose gel electrophoresis Agarose slab gels (13x 14x0.5 cm) 0.8% were made up in electrode buffer (Tris base, 1.08%; NaEDTA, 0.093% and boric acid 0.55% (pH 8.0)). Gels were either run for 3 hr at 60 mA and approximately 80 V or overnight at 12 mA and 20 V, stained in ethidium bromide (1.0 ug/ml) for 30 min and then examined on a Chromoto-Vue transilluminator (model C62, Ultra Violet Products Inc.).

Gels were photographed using Kodak Pan Lithographic film and Wratten (No 1 5) and orange filters.

To detect smaller DNA fragments, 1.4% agarose gels made up in Tris/acetate buffer (20.0 mM, pH 7.7) containing 2.0 mM EDTA. The gels were run at 60 mA for 3-3.5 hr after which time the bromophenol blue indicator dye had moved 75% the length of the gel.

K99 detection and assay Colonies which were positive for K99 antigen were normally detected by slide agglutination.

K99 antigen was assayed by a back titration procedure in which bacterial cultures were serially diluted in 0.5 ml of form (0.25%) saline in small glass tube (8x0.7 (diam) cm). Half a millilitre of a standard anti-K99 antiserum was then added to each tube and, after thorough mixing, the tubes were incubated for 60 min at 370C. The tubes were then centrifuged, 50 l of the supernatant from each tube were placed in U-shaped wells in a plastic tray and 50 ul of a standard suspension of K99 positive E. coil were finally added to each well. The plastic trays were then wrapped in a plastic bag to prevent dehydration and incubated at 370C overnight.Where the bacterial suspension under test had adsorbed all the anti-K99 antiserum, the standard suspension of K99 positive E. coil formed a compact 'button' on the bottom of the well. Conversely, in the absence of complete absorption the remaining anti-K99 antibody agglutinated the test suspension and prevented 'button' formation.

Copy number determination To determine plasmid copy number, E. coli C transformants were grown in 20.0 ml of M9 medium (Na2HPO4, 0.6%; KH2PO4, 0.3%; NaCI, 0.05%; NH4CI, 0.1% and CaCI2 added to 0.1 mM) containing 0.2% glucose and 0.5% casamino acids (Difco) at 370C and 140 cycles per min. When the cell concentration had reached OD650 of 0.1, methyl tritiated thymidine was added to a final concentration of 1.0 uCi/ml as well as unlabetled thymidine to 2.0 ug/ml and deoxyadenosine to 200 ug/ml (Boyce and Setlow, Biochim. Biophys. Acta 61.618--620, (1962)).The cultures were then incubated overnight at 37 OC, harvested by centrifugation (10 000 g, 10 min at 40C) and washed twice with 20.0 ml volumes of TES buffer (Tris, 50 mM: NaCI, 50 mM; EDTA 5 mM; pH 8.0). Cells pellets were then resuspended in 0.1 ml of 20.0% sucrose to which 40 ul lysozyme (5 mg/ml) and 60 ul of RNase (1 mg/ml) were added. The cells were incubated at 370C for 30 min after which cell lysis was accomplished by adding 200 ul of Brij lysis mixture, incubating for a further 30 min at 370C and then shearing the cells through a 25 gauge needle.

The lysates were made up to 8.0 ml with TES buffer and then 8.0 g of CsCI and 0.2 ml of ethidium bromide (10 mg/ml) were added. The lysates were centrifuged at 40 000 rpm for a minimum of 40 hr at 1 90C, after which holes were punched in the bottom of the centrifuge tubes and fractions (0.3 ml) were collected. Ten microlitre aliquots of each fraction were placed on filter paper squares (1.5 x 1.5 cm) which were dried, treated twice with cold TCA (5%), washed with ethanol and dried again. Each filter paper square was placed in a scintillation vial, 5.0 ml of scintillation fluid (Koch Light, KL502) was added and the vials counted on a Beckman scintillation counter.

Results The results obtained are illustrated by reference to the accompanying drawings, in which: Figure 1 shows agarose gels (0.8%) illustrating the result of the following restriction enzyme digests of the hybrid plasmids pTP13-53: A (a) undigested pTP 13; (b) Hind Ill digest of pTP 13; (c) Hind Ill digest of pTP23; (d) Hind III digest of pTP33; (e) Hind Ill digest of pTP43; (f) Hind Ill digest of pTP53; (g) Sal 1 digest of pTP 13; (h) Sal 1 digest of pTP23; (i) Sal 1 digest of pTP33: (j) Sal 1 digest of pTP43; (k) Sal 1 digest of pTP53; (I) Eco R1 digest of -DNA; B (a) undigested pTP1 3; (b) Bam H1 digest of pTP1 3; (c) Bam H1 digest of pTP23; (d) Bam H1 digest of pTP33; (e) Bam H1 digest of pTP43; (f)Bam H1 digest of pTP53; (g)Eco R1 digest of pTP13; (h) Eco R1 digest of pTP23; (i) Eco R1 digest of pTP33; (j) Eco R1 digest of pTP43; (k) Eco R1 digest of pTP53; (I) Eco R1 digest of -DNA; C (a) Pst 1 digest of pTP 13; (b) Pst 1 digest of pTP23; (c) Pst 1 digest of pTP33; (d) Pst 1 digest of pTP43; (e) Pst 1 digest of pTP53; (f) Eco R1 digest of -DNA; (g) Bam H1 digest of pTP1 1; (h) Bam H 1 of pTP 12; (i) Pst 1 digest of pBR322; and (k) Eco R 1 digest of -DNA; Figure 2 shows agarose gels (0.8%) illustrating the results of the following restriction enzyme digests of those plasmids derived from pTP 13 and pTP43 either by double digestion followed by religation orsubcloning a Pst 1 fragment of pTP 13 and pTP43: A (a) Hind III/Sal 1 digests of pTP 1 3; (b) Hind Ill/Sal 1 digest of pTP 131S; (c) Hind Ill/Sal 1 digest of pTP 132S; (d) Hind ill/Sal 1 digest of pTP43; (e) Hind III/Sal 1 digest of pTP431 S; (f) Hind III/Sal 1 digest of pTP432S; (g) Eco R1/Hind Ill digest of pTP 13; (h) Eco R1/Hind Ill digest of pTP 131 E; (i) Eco R1/Hind Ill digest of pTP 1 32E; (j) Eco R1/Hind Ill digest of pTP43; (k) Eco R1/Hind Ill digest of pTP431E; Eco R1/Hind III digest of pTP432E; B (a) Pst 1 digest of pTP 13; (b) Pst 1 digest of pTP 1 31; (c) Pst 1 digest of pTP 132; (d) Pst 1 digest of pTP43; (e) Pst 1 digest of pTP432; (f) Pst 1 digest of pTP433; (g) Bam H1/Hind Ill digest of pTP13; (h) Bam H 1/Hind Ill digest of pTP 131 B; (i) Bam H 1/Hind Ill digest of pTP 132B; (j) Bam H 1/Hind Ill digest of pTP43; (k) Bam H1/Hind Ill digest of pTP431 B; (I) Bam H1/Hind Ill digest of pTP432B; C (a) Bam H1 digest of pTP 131; (b) Bam H1 digest of pTP 132; (c) Bam H1 digest of pTP432; (d) Bam H1 digest of pTP433; (e) Eco R1 digest of -DNA; (f) undigested pTP131;(g) undigested pTP432.

D (a) Sal 1 digest of pTP 131; (b) Sal 1 digest of pTP 1 32; (c) Sal 1 digest of pTP432; (d) Sal 1 digest of pTP433; (e) Eco R1 digest of -DNA; (f) undigested pTP131; (g) undigested pTP432; (h) Eco R1 digest of pTP 131; (i) Eco R1 digest of pTP 132; (j) Eco R1 digest of pTP432; and (k) Eco R 1 digest of pTP433; and Figure 3 shows restriction enzyme maps of the hybrid plasmids which code for the K99 antigen.

The heavy lines represent pBR322 and the figures indicate the size of the fragments in kilobases. A is pTP 13, B is pTP43, C is pTP432, D is pTP 131 and pTP433.

Cloning of DNA coding for the K99 antigen Plasmid DNA prepared from E. coli K12 which contained a large, conjugative plasmid coding for the K99 antigen (designated pTM 1) was digested with Hind Ill restriction enzyme. The vector plasmid pBR322 was also digested with Hind Ill as well as alkaline phosphatase to remove terminal phosphate groups to prevent religation without fragment incorporation.

Vector plasmid and the Hind Ill digest were ligated after ATP, dithiothreitol and T4 DNA ligase addition. Competent cells of E. collC were then transformed with this recombinant DNA preparation and amplicillin resistant transformants were screened for K99 formation by slide agglutination. Plasmid DNA was prepared from five transformants which were K99 positive and tetracycline sensitive in order to characterise the DNA fragments which had been cloned into pBR322. The five hybrid plasmids were designated pTP 1 3-pTPS3.

The results shown in Figure 1A indicate that all five DNA preparations yielded inter alia the same two fragments on Hind Ill digestion. One of these was linear pBR322 and the other, a 13.1 kb fragment of pTM 1. Digestion of the hybrid plasmids with Sal 1 , Eco R1, Bam H1 and Pst 1 all indicated that the orientation of the cloned fragments were identical except for pTP43 (Figure 1 B and 1 C).As the expression of K99 in E. collC (pTP43) appeared, on the basis of slide agglutination, to be as good as expression in any of the other E. coil C strains containing the other hybrid plasmids, there was a clear indication that the expression of K99 was independent of the orientation of the K99 genetic determinants with respect to the vector plasmid. The digestion data also indicated that the inserted fragments had only one Eco R1 and one Sal 1 site but at least five Bam H1 and five Pst 1 sites. These results are summarised in the restriction enzyme maps for pTP 13 and pTP43 shown in Figures 3A and 3B respectively.

Sub-cloning of the K99 genetic determinants As the Hind Ill fragment was large (13.1 kb) in comparison to the vector plasmid and evidently contained sites for Bam H 1, Sal 1, Eco R1 and Pst 1, it was decided to attempt isolation of a smaller fragment in the hope that the resulting hybrid plasmid would have a higher copy number and, therefore, possibly give a higher level of K99 expression. The plasmids pTP 1 3 and pTP43 were simultaneously digested with Hind Ill and either Sal 1, Bam He, or Eco RI.After inactivation of the restriction enzymes the resulting digests were rn-ligated and the DNA preparations then used to transform competent cells of E. coli C. As before, ampicillin resistant transformants were tested for K99 formation by slide agglutination. In all cases it was possible to isolate K99 positive transformants.

Plasmids derived from Hind Ill/Eco R1 digest of pTP13 were designated pTP 131 E, pTP 1 32E etc and those derived from pTP43 were designated pTP431 E, pTP432E etc. Similarly plasmids derived from Hind I I I/Bam H1 digests were designated pTP 1 31 B, pTP 132B, pTP43 1 B and pTP432B and those derived from Hind Ill/Sal 1 digests were designated pTP 131S, pTP 132S, pTP431S and pTP432S.

Restriction enzyme digests of plasmid DNA prepared from these strains revealed that, in some cases, the pattern of bands were identical to that of the parent hybrid plasmid, but, in other cases, certain fragments had been eliminated (Figure 2A). Only those hybrid plasmids which were known to be smaller than the parent hybrid plasmids (pTP 1 3 and pTP43) were retained for further studies on copy number estimation and K99 expression.

Because the Pst 1 site in pBR322 occurs in the ampicillin resistance gene (unlike the Bam H1, Sal 1 and Hind Ill sites which all occur in the tetracycline resistance gene) it was not possible to adopt the strategy of double digestion with Pst 1 and Hind Ill as this would have destroyed resistance to both tetracycline and ampicillin. Consequently, Pst 1 digests of both pTP 13 and pTP43 were mixed with pBR322 which had been digested with Pst 1 and treated with alkaline phosphatase and the resulting mixture was ligated. Competent cells of E. coli C were transformed with this DNA preparation and tetracycline resistant colonies were screened for K99 production. Four K99 positive transformants were found; two (designated pTP 131 and pTP 132) contained hybrid plasmids with Pst 1 fragments from pTP 1 3 and the other two (designated pTP432 and pTP433) contained Pst 1 fragments from pTP43. Plasmid DNA was prepared from the E. coli C strains containing pTP 131,132,432 and 433 and digested with Bam H1, Eco R1 and Sal 1 (Figure 2B) as well as Pst 1. The Pst 1 digests revealed that all four hybrid plasmids contained one 6.3 Kb fragment inserted into the Pst 1 site of pBR322. The hybrid plasmids contained one Eco R1 and one Sal 1 site as well as three Bam H1 sites.As pBR322 is known to contain one Eco R1, one Sal 1 and one Bam H1 site, it was concluded that the inserted fragment had no Eco R1 or Sal 1 site but two Bam H1 sites. From the Bam H1 digest (Figure 2E it was also apparent that the orientation of the fragment was identical in pTP 1 31 and pTP433 but opposite in pTP432. The restriction enzyme maps for pTP 131, pTP432 and pTP433 are shown in Figures 3C and 3D of the accompanying drawings. The results obtained with pTP 132 could not be readily interpreted and after several subcultures the strain containing this plasmid failed to produce K99.

Copy number determinants of hybrid plasmids Copy number estimations were carried out on pBR322, pTP 13, pTP43, pTP 131, pTP 132, pTP432 and pTP433 as described in the earlier section headed "Copy Number Determination". The results given in the Table below show that the copy number of pTP 13 and pTP43 were roughly one third that of pBR322. Estimations of pTP 132 and pTP433 repeatedly indicated that the number of copies was similar to those of pTP 13 and pTP43, but the number of copies of pTP 131 and pTP432 were significantly higher.

The expression of the K99 pilus antigen In order to compare the level of K99 antigen expressed by the original plasmid isolated from E.

coil K12 (pTM 1) with the levels expressed by the hybrid plasmids containing the K99 genetic determinants, it was necessary to transfer the plasmid pTM I into E. coli C. This was of added importance because unlike E. coli K12 the K99 formed by E. coli C does not deteriorate in stationary phase when cultured in Trypticase Soy Broth (Isaacson, lnf. and Immun. 28 190194 (1980)).

Consequently, E. coli K12 (pTM 1) was transformed with pBR322 and one of the resulting transformants was mated with a strain of E. coli C which was resistant to high levels of rifampicin.

Exconjugants of E. coli C which had received both pTM 1 and pBR322 (by co-migration) were selected by plating on agar containing both ampicillin (50 ug/ml) and rifampicin (100 ug/ml) and further characterised by their requirement for methionine when grown on a minimal medium.

The E. coli C strains containing the various hybrid plasmids as well as pTM 1 were cultured in Tryticase Soy Broth and then assayed for the K99 antigen as described above. The results are given in the following Table.

Table The production of K99 pilus antigen by strains of E. coli C containing K99 hybrid plasmids when grown in Trypticase Soy Broth to stationary phase.

OD650 overnight Plasmid Mol. Wt Plasmid culture K99 (unit/mI) K99/OD650 Copy No (MD) pTM 1 +pBR322 3.7 11.4-17.1 3.1-4.6 - 2 pTP13 4.4 384.4-576.7 87.4-131.0 22 11.12 pTP43 4.3 384.4-576.7 89.4-134.0 25 11.12 pTP131 4.1 171.0-256.3 41.7-62.5 59 6.7 pTP432 3.6 171.0-256.3 47.5-71.2 51 6.7 pTP433 4.0 144.0-1 71.0 28.9-43.3 29 6.7 pTP 131 E 4.1 384.4-576.7 93.8-i 40.7 - pTP431E 4.5 384.4-576.7 85.4-128.2 - pTP43iS 3.1 50.6-76.0 16.3-24.5 - pTP131B 4.1 256.3-384.4 62.5-93.8 - pTP431B 5.0 171.0-256.3 34.2-51.3 - 'Copies per chromosome-mean of two or three determinations.

2The average copy number estimation of pBR322 was 76.

It was found that the highest levels of K99 expression were given by E. coli C (pTP 13) and E. coli C (pTP43) which produced almost thirty times as much K99 antigen asE. coli C (pTMi).E. coli C containing pTP 131, 132, 432, or 433 failed to produce as much K99 antigen as either E. coli C (pTP 13) or E. coli C (pTP43) even though the number of copies of pTP 131 and pTP432 were significantly higher than pTP 13 or pTP43.

Expression of the K99 antigen by the plasmids pTP 131 E and pTP431 E which were made by ligation of pTP 13 and 43 after digestion with both Hind Ill and Eco Tri, gave results as high as pTP 13.

The other plasmids, pTP431S (formed by ligation of a Sal 1/Hind Ell digest of pTP43) and pTP 131 B and pTP431 B (formed by ligation of Bam H1/Hind ill digests of pTP 13 and 43 respectively) failed to produce as much K99 antigen as pTP 13 or pTP43. In this respect they appear to be similar to pTP 131, pTP432 and pTP433.

Discussion In this example, a 13.1 Kb fragment of the plasmid pTM 1 which codes for the K99 antigen was cloned into the multicopy plasmid pBR322. Restriction enzyme analysis of the hybrid plasmids containing this fragment indicated that four or five (pTP 3, pTP23, pTP33 and pTP53) were identical while the remaining plasmid (pTP43) had the DNA fragment in the opposite orientation with respect to the cloning vector. Examination of the levels of K99 antigen production by E. coli C strains containing pTP 13 and pTP43 demonstrated that the expression of K99 was independent of orientation (Table).

This result was in contrast to previous work on the K88ab genetic determinants where Hind Ill fragment of plasmid DNA isolated from all six K88ab positive clones were found to be in the same orientation. Mooi et al (loc. cit) had also found that K88ab expression from an 11.8 Kb Hind Ill fragment cloned into pBR322 was highly orientation dependent.

The expression of K99 by E. coli C (pTP 13) or E. coli C (pTP43) was approximately thirty times the level produced by E. coli C (pTPM 1). This correlates well with the copy numbers of pTP 13 and pTP43 which were high in relation to pTM 1 (the low copy, K99 plasmid from which the 13.1 Kb Hind Ill fragment was originally obtained) but only about a third of the copy number of pBR322.

Claims

Claims

1. A recombinant DNA molecule comprising a nucleotide sequence substantially coding for a filamentous pilus antigen of an enteropathogenic strain of Escherichia coli.

2. A recombinant DNA molecule comprising a nucleotide sequence substantially coding for the K99 antigen of an enteropathogenic strain of E. coli.

3. A recombinant DNA molecule according to claim 1 or 2, which molecule comprises one or more expression control sequences.

4. A recombinant DNA molecule according to claim 3, wherein the or at least one of the expression control sequences is positioned adjacent to the nucleotide sequence substantially coding for the antigen.

5. A recombinant DNA molecule according to claim 3 or 4, wherein the or at least one of the control sequences is derived from E. coli DNA.

6. A recombinant DNA molecule according to claim 3 or 4, wherein the or at least one of the control sequences is derived from a source that is heterologous toE. coli.

7. A recombinant DNA molecule according to any one of claims 1 to 6, wherein the nucleotide sequence substantially coding for the antigen is present in a cloning vehicle.

8. A recombinant DNA molecule according to claim 7, wherein the cloning vehicle is a plasmid having a relatively high copy number and/or a relatively low molecular weight compared with a parent plasmid from which the nucleotide sequence substantially coding for the antigen may be derived.

9. A recombinant DNA molecule according to claim 7 or 8, wherein the cloning vehicle is the plasmid pBR322.

1 0. A host cell containing a recombinant DNA molecule substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coli.

it. A method of preparing a recombinant DNA molecule substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coli, which method comprises inserting a nucleotide sequence substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coli into a cloning vehicle.

1 2. A method according to claim ii, wherein the nucleotide sequence is obtained by digestion with a restriction enzyme of a plasmid containing the desired sequence.

13. A method according to claim 12, wherein the plasmid is the pTM 1 plasmid obtainable from E.

coilK12.

14. A method according to claim ii or 12, wherein the restriction enzyme is Hind Ill.

1 5. A hybrid plasmid identified by the code pTP13, pTP23, pTP33, pTP43 or pTP53.

16. A hybrid plasmid identified by the code pTP13 or pTP43.

1 7. A proteinaceous expression product of a recombinant DNA molecule according to any one of claims 1 to 9, 1 5 and 16.

1 8. A vaccine comprising a proteinaceous expression product according to claim 1 7.

1 9. A recombinant DNA molecule comprising a nucleotide sequence substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coli substantially as hereinbefore described with reference to the Example.

20. A host cell containing a recombinant DNA molecule substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coil substantially as hereinbefor described with reference to the Example.

21. A method of preparing a recombinant DNA molecule substantially coding for a filamentous pilus antigen of an enteropathogenic strain of E. coli substantially as hereinbefore described with reference to the Example.