Methods of identifying antigen gene sequences

Details Methods of identifying antigen gene sequences Methods of identifying antigen gene sequences

2001-05-21

2004-07-06

Fredman, Jeffrey (Department: 1637)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S820000, C530S825000, C514S002600

Reexamination Certificate

active

06759516

ABSTRACT:

The present invention relates to methods of identifying gene sequences of potential vaccine antigens. Also included are gene sequences and the polypeptides encoded by the gene sequences as well as the use of such sequences to induce a protective immune response in animals. Particularly, the invention relates to identifying potential antigen gene sequences of Mycoplasma, preferably
Mycoplasma hyopneumoniae.
INTRODUCTION
The success of a vaccine to a pathogen resides in identifying a suitable antigen of a pathogen which is readily accessible to a host immune system. Once identified, it can form the basis for protection against the pathogen.
In recent years a number of vaccines have been commercialised by Animal Health companies. Many of the vaccines are based on inactivated whole cell bacterins. Although these vaccines provide a reasonable level of efficacy there is considerable scope for improvement. Therefore the presently available vaccines could be improved on by developing vaccines that were not based on whole cells or fractions of whole cells.
Other means of generating vaccines against pathogens includes the use of crude inactivated antigen mixtures of various proteins. However, the problem with these methods is that they present to the host a variety of antigens, none of which may provide suitable overall protection since the proteins most readily accessible to a host immune system are swamped by the other antigens. Moreover, some pathogens are difficult or expensive to grow because of their fastidious requirements. Some pathogens are also harmful to handle. Therefore, a vaccine which does not require the growing of the cells or bacteria and the processing of whole cells or fractions of whole cells would provide a safer, cheaper and possibly more efficacious vaccine.
One way of achieving a cleaner and more specific vaccine is by using recombinant protective antigens. By providing specific antigens, only those antigens readily accessible to the host immune system may be used.
There is the problem of identifying potential antigens. One way is to create a gene library. However, from the library, it is very time consuming to determine those sequences which may include a potential protective antigen.
To efficiently screen through a large number of potential antigen genes for their efficacy in providing some level of protection from disease it is useful to initially clone them as expressing clones. In this way the need for tedious and time consuming clone analysis and subsequent expression subcloning is avoided. The whole process of antigen discovery is speeded up. Applicants now provide a novel method to ensure that the clones initially investigated were expressing recombinant protein.
Previously, expression libraries have been screened for clones expressing all or part of a specific protein by using an antibody specific for or enriched for the protein of interest.
Identifying the DNA sequences that code for the proteins, also makes it possible, using appropriate expression vehicles, to form recombinant DNA molecules and to transform appropriate hosts (eg., prokaryotic or eukaryotic hosts) with those recombinant DNA molecules. Culturing of the transformed hosts then permits the hosts to express the DNA sequences and to produce the desired proteins.
Administering the produced and subsequently isolated proteins, active ingredients or combinations thereof (eg., by injection), in an amount sufficient to elicit a protective immune response, provides a means for immunising against infections.
One pathogen for which vaccines have been commercialised by Animal Health companies is
Mycoplasma hyopneumoniae
. This pathogen causes
Enzootic pneumonia
in pigs. It rarely causes death, but often results in severe morbidity and reduced performance manifesting in significant depression in feed conversion efficiency resulting in reduced weight gain in pigs. The animals show symptoms of coughing and fever and are often prone to secondary infection by opportunistic microorganisms.
Numerous attempts to provide a vaccine against
Mycoplasina hyopneumoniae
have not been terribly successful. Particularly, the use of heat inactivated, live or extract of Mycoplasma have proven to be ineffective in providing protection. Some vaccines based on inactivated whole cell bacterins have shown some level of efficacy but there is scope for improvement.
Additionally,
Mycoplasma hyopneumoniae
is difficult and expensive to grow because of its fastidious requirements. Therefore the presently available vaccines could be improved on by developing vaccines that were not based on whole cells or fractions of whole cells.
Accordingly, it is an object of the present invention to overcome or at least alleviate some of the problems of the prior art.
SUMMARY OF THE INVENTION
In one aspect of the present invention there is provided a method of identifying expression proteins translated from a nucleotide sequence in an expression vector, said method comprising the use of a marker co-expressed with a protein translated from the nucleotide sequence.
In a preferred embodiment of the present invention there is provided a use of a polyHis tag for identifying expression proteins encoded by a nucleotide sequence in an expression vector wherein said polyHis tag is co-expressed with said protein.
In another aspect of the present invention there is provided a method for identifying expression proteins encoded by a nucleotide sequence from a mixture of nucleotide sequences, said method comprising the steps of:
providing an expression vector including a marker;
introducing a nucleotide sequence from the mixture into the expression vector; and
identifying an expression protein expressed by the expression vector by determining the presence of a fusion protein comprising the marker co-expressed with the expression protein.
In a preferred embodiment the marker is a polyHis tag.
In a further preferred embodiment, the expression vector is transfected into a host cell for expression of a marker-fusion protein comprising an expression protein. Preferably the marker fusion protein is a polyHis tag-fusion protein comprising a polyHis tag and an expression protein. Introduction of a population of expression vectors into the host cell may create a genomic or cDNA library which may be screened.
In a further preferred embodiment of the method there is included a step of purifying the expression protein using the marker expressed in the fusion protein. Preferably the marker is a polyHis tag.
There is provided in another aspect, a purified recombinant protein identified according to the above method. Preferably the recombinant protein is an expression protein.
In another aspect, there is provided a purified nucleotide sequence which encodes a recombinant protein identified according to the above method.
In another aspect of the present invention there is provided an expression vector including a marker, preferably a polyHis tag for use in identifying expression proteins encoded by a nucleotide sequence.
In yet another aspect of the invention, there is provided a host including an expression vector, said vector including a marker, preferably a polyHis tag for use in identifying an expression protein.
In a further aspect of the present invention, there is provided a method of identifying gene sequences encoding antigenic proteins from a sample of nucleotide sequences, said method comprising the steps of:
providing an expression vector including a marker;
introducing a nucleotide sequence from the sample into the expression vector;
identifying an antigenic expression protein expressed by the expression vector by determining the presence of a fusion protein comprising a marker co-expressed with the expression protein translated by the nucleotide sequence; and
identifying the antigenic expression protein.
Preferably the marker is a polyHis tag.
In a preferred embodiment, the method further includes determining the gene sequence of the antigenic protein encoded by the expression vector.
In another aspect of the invention there is provided a method of sc

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