DNA molecule encoding gonorrhoeal hybrid PIA/PIB protein

Details DNA molecule encoding gonorrhoeal hybrid PIA/PIB protein DNA molecule encoding gonorrhoeal hybrid PIA/PIB protein

1999-06-15

2002-02-19

Devi, S. (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069700, C435S069100, C435S320100, C435S252300, C536S023700, C536S023400, C536S023100, C530S350000, C530S300000, C424S249100, C424S190100, C424S192100

Reexamination Certificate

active

06348332

ABSTRACT:

TABLE OF CONTENTS
1. Introduction
2. Background of the Invention
2.1. Vaccines
2.2. Gonococcal Antigens
2.2.1. Gonococcal Protein I
2.3. Diagnostic Probes
3. Summary of the Invention
4. Description of Figures
5. Description of the Invention
5.1. Identification and Isolation and Sequence of the PIA Gene
5.2. Identification, Isolation and Sequence of the PIB Gene
5.3. Production of PIA, PIB or PIA/B Hybrids by Recombinant DNA Techniques
5.3.1. Preparation of a PIA Gene Cloning/Expression Vector
5.4. Identification and Purification of the PIA and PIB Gene Products
5.5. Preparation of PIA/B Hybrids
5.6. Production of PIA, PIB and Hybrid Proteins by Synthetic Techniques
5.7. Formulation of a Vaccine
6. Example I: PIA Gene Identification Isolation and Expression
6.1. General Procedures Used for Preparation of the Plasmids
6.1.1. Conditions for Restriction Enzyme
6.1.2. Restriction Enzyme Buffers
6.1.3. Identification of Relevant Restriction Fragments
6.1.4. Cloning of Fragments
6.1.5. Subcloning and Sequencing of Fragments
6.2. Gene Expression
7. Example 2: Identification, Isolation, Sequencing and Expression of PIB Gene and Construction and Expression of PIA/B Hybrids
7.1. Insertion of a Selectable Marker into the PI Gene
7.2. Cloning, Sequencing and Expression of the PIB Gene
7.3. Construction and Analysis of PIA/B Hybrid Strains
7.4. Discussion
8. Deposit of Microorganisms
1. INTRODUCTION
Gonorrhea is at the present time one of the most widespread venereal disease worldwide, with several million cases occurring in the United States alone each year. The causative agent of the disease is the gonococcus
Neisseria gonorrhoeae,
a bacterium which has throughout its history developed resistance both to traditional antibiotic treatment, and to some extent to the bactericidal activity of normal human serum. The present inability to control the infection by traditional means has made the development of a vaccine which can effectively prevent the infection of utmost importance. The present invention provides a DNA sequence coding for a specific
N. gonorrhoeae
outer membrane protein; the cloned product of this particular DNA sequence provides a suitable basis for such a vaccine. The present invention also provides species-specific oligonucleotide sequences useful as diagnostic probes for the detection of gonorrheal infection.
2. BACKGROUND OF THE INVENTION
2.1. Vaccines
The production of a protective immune response against any given infection agent in vertebrates depends initially on the provision of the appropriate stimulus to the host's immune system. The infectious organism itself typically provides numerous stimulatory compounds, or antigens, by the very nature of its cell membrane composition, or by the metabolic products it releases in the host's body. These substances, usually larger molecules such as proteins, lipopolysaccharides or glycoproteins, are recognized by the immune system as foreign, and provoke one or more different types of reaction from the host in an effort to remove or disable the invading organism. The antigen may cause production of sensitized lymphocytes (T-cells) which provide a cell-mediated immunity. Alternatively, an antigen may stimulate the synthesis and release of free antibody into the blood and other bodily fluids (humoral immunity). The development of the body's protective immune response depends upon achieving a threshold level of stimulation of one or both of these systems.
A temporary immunity against infection can in many cases be provided by giving an individual preformed antibodies from another individual of the same or different species. This is known as passive immunity. One example of such immunity is the protection afforded to a fetus and newborn by placental transfer of maternal antibodies, as well as transfer of antibodies through milk. Another example is the pooled adult gamma globulin frequently used to prevent or modify the effects of exposure to measles, chicken pox, hepatitis, smallpox and tetanus. These acquired antibodies, however, are gradually utilized by interaction with the antigen or catabolized by the body, and thus the protection is eventually lost.
A more permanent form of protection is afforded by active immunization. Vaccination confers an active protective immunity by employing a harmless or nonvirulent form of the antigen (e.g., a killed or genetically altered bacterium, or an isolated glycoprotein from the cell wall) as a primary stimulus to the immune system. This provokes a rather slow response in antibody production which peaks and falls off. However, the body has been alerted to the existence of the antigen, and the next time exposure-occurs, presumably with the live, virulent organism, a secondary response, with a much more rapid and abundant production of antibodies is observed. This secondary response will typically be sufficient to prevent the microorganism from establishing itself sufficiently to be able to cause a full-blown infection.
A vaccine may take a variety of forms, some of which are more effective than others in conferring the protective effect desired. Historically many vaccines have been prepared by killing or inactivating the microorganism causing the disease of interest, and using the killed cells as the active immunogenic agent. Vaccines of this type have been used against typhoid, cholera and poliomyelitis (Salk vaccine). The problem with this type of vaccine is that the treatment required to kill the microorganisms, such as formaldehyde, may frequently alter or destroy the microbe's useful antigens, and thus poor or incomplete immunity may be obtained.
An alternate form of vaccine is that which employs attenuated microorganisms. An attenuated microorganism is one which is still living, and capable of multiplying in the body after administration, but which has either been modified in some way to render it avirulent, or else is a strain which is virulent in other microorganisms, but avirulent in man. The advantage obtained by using an attenuated organism is that the attenuation usually does not affect its antigenicity, thereby providing a more efficient stimulus to the immune system. Attenuated vaccines for measles, rubella and poliomyelitis (Sabin vaccine) have achieved widespread use. Attenuation is typically achieved by altering the growth conditions of the microorganism, or, more recently, genetically modifying the organism's virulence. Although generally more effective than killed vaccines, however, there is danger involved in the possibility of the live organisms reverting to virulence, thereby causing disease symptoms.
Because of the problems associated with whole organism vaccines, it has become more common in recent years to employ individual protective antigens as the active agent in vaccine compositions. Although isolation and identification of the particular antigens of an organism which do stimulate a protective response is not always simple, once identified, this method provides an effective alternative to the whole organism vaccines, without the attendant disadvantages.
Examples of types of antigens which are typically useful for this purpose are purified cell or capsid components, such as bacterial toxins (which must be detoxified to yield a toxoid), bacterial or viral toxin subunits, cell wall polysaccharides, or capsid glycoproteins. These components are often highly effective in producing immunity, but difficulties arise in the actual purification. It is critical that the antigen of interest be isolated more or less completely from extraneous cellular materials, the presence of which can frequently cause an adverse immunologic or metabolic response concurrent with the desired immunity producing reaction. The purification processes required are frequently complex, tedious and prohibitively expensive and the results not always completely predictable. This problem can in some case be avoided by synthetic preparation of small peptide sequences which correspond to epitopes on a microbial antigen. There are, in some circumstances, drawbacks to this techniqu

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