PRODUCTION OF IMMUNOREACTIVE PROTEINS BY USING THE METHODS OF RECOMBINANT DNA TECHNOLOGY THAT USABLE FOR SEROLOGICAL DIAGNOSIS OF BABESIA OVIS INFECTION

TECHNICAL FIELD

Invention relates to the production of immunoreactive proteins obtained from Babesia ovis organisms by using the methods of recombinant DNA technology. These immunoreactive proteins may be used as antigens in a diagnostic kit which enables to detect quantitatively the specific-antibodies in the field infections.

PRIOR ART

Tick-borne parasitic diseases are preeminent health and management problems of livestock. Ovine babesiosis is a primary tick-borne disease caused by apicomplexan parasites Babesia ovis, B. motasi and B. crassa in Europe, Africa, Asia, and the Far East. Babesia ovis, the main causative agent of ovine babesiosis, causes clinical signs characterized by fever, hemolytic anemia, hemoglobinuria, jaundice, weakness, anorexia, and mortality in small ruminants. The clinical cases are usually severe. Babesia ovis is highly pathogenic, causing pancytopenia, characterized by anemia, thrombocytopenia and leukopenia in severe cases. Untreated cases end up in death of sick animals, and some animals die as a result of heavy infection, despite treatment. Many recurrences also occur after treatment. Compensation of the abnormalities in the hematological picture takes a long time in the animals treated with anti- babesial drugs. For these all reasons, the disease has considerably economic importance in terms of death cases, yield losses and the costs of treatment in the livestock industry.

The current control of ovine babesiosis is based only on chemotherapy and limited tick control measures. Imidocarb dipropionate (IMDP) has been used in the treatment of the disease, has also chemoprophylactic effectiveness against ovine babesiosis, with certain duration and rates. However the strength and duration of protection provided by chemoprophylactic drugs is less, and the other problems are related with the side effects of the drug and the residues in the organs and milk of drug-treated animals. Tick-control measurements in sheep are difficult because they graze on pastures during long periods of the year and expose to tick infestations. In the areas where the endemic status of B. ovis is unstable, an immunization program is needed. However, immunoprophylaxis is lack for the ovine Babesia species. An available vaccine would reduce the losses in outbreaks of the disease; therefore, the development of vaccines is crucial.

The diagnosis of acute babesiosis is based on the clinical signs of the disease and on the demonstration of the parasites in the erythrocytes in Giemsa-stained blood films by microscopy. Microscopic and clinical examinations are disqualified for the diagnosis of the subclinically infected carrier animals. The carrier animals have been determined by the immunological and molecular methods. Although there is no commercial product used for serological diagnosis of B. ovis infections, indirect fluorescent antibody test using the blood of infected animal as antigens have implemented in some laboratories. In addition, synthetically derived Babesia bovis molecules were used as antigen in ELISA in some studies.

With the importance of immunoreactive recombinant proteins in the diagnosis of the diseases and in the production of recombinant vaccines, many studies have been conducted in order to obtain recombinant proteins from different Babesia species. These proteins are valuable sources in the specific diagnosis of the disease as well as vaccine candidates for protection against the disease. Using the technique of gene cloning, it is possible to isolate parasite genes encoding a specific protein and to introduce these genes into bacteria, where these proteins can be expressed in large quantities as recombinant antigens. The cDNA libraries are the most valuable sources for detection of the genes encoding the immunoreactive proteins.

In recently DNA-based methods which are specific and sensitive to detect parasites in animals and vectors have been used to identify vector species and to genotype parasites. These methods are valuable research tools for specific studies. But these are time-consuming and costly for examination of large number of samples. Therefore, it is needed to develop diagnostic methods which are specific, sensitive, cost-effective and even be applied in the field for implementation of effective control strategies against the tick-borne diseases. The parasite antigens prepared from infected blood contains some of the host proteins. Therefore, false-positive reactions are encountered in the serological tests using these antigens. Immunoreactive proteins obtained by recombinant DNA technology do not include host factors, for this reason the specificity and sensitivity of the tests using these proteins are usually very high. These proteins are able to synthesize with desire amount and time in the laboratory. They are conveniently used as antigens in a quantitative assay such as ELISA or in the immunochromotography test applied even in the field. Immunoreactive proteins are also of significant importance as the vaccine candidates in the prevention of diseases.

The immunoreactive proteins of some Babesia species have commonly been used as diagnostic antigens in the diagnosis of equine, bovine and canine babesiosis. There is no study about the producing of immunoreactive recombinant proteins of B. ovis. BRIEF EXPLANATION OF INVENTION

Invention relates to the production of immunoreactive proteins obtained from Babesia ovis organisms by using the methods of recombinant DNA technology. The specific genes encoding these proteins were detected with immunoscreening of cDNA library constructed from mRNA of Babesia ovis. These specific genes were cloned in the pGEX expression vectors, and then recombinant vectors were transformed to the competent E. coli cells for expression of recombinant proteins. The proteins were expressed in E. coli cells as GST-fusion proteins. DETAILED EXPLANATION OF INVENTION

Invention relates to the production of immunoreactive proteins obtained from Babesia ovis organisms by using the methods of recombinant DNA technology. The specific genes encoding these proteins were detected with immunoscreening of cDNA library constructed from mRNA of Babesia ovis. These specific genes were cloned in the pGEX expression vectors, and then recombinant vectors were transformed to the competent E. coli cells for expression of recombinant proteins. The proteins were expressed in E. coli cells as GST-fusion proteins.

These GST-fusion proteins were purified by glutathione affinity chromatography. The antigenicity of the purified proteins was tested in an Enzyme Linked Immunosorbent Assay (ELISA) by using immune sera from lambs experimentally infected with B. ovis. The results of ELISA showed that the two immunoreactive proteins detected B. ow ^' s-specific antibodies in the eighth day of experimental infection. Therefore, these proteins would be used as antigens on the preparation of a diagnostic kit for serological diagnosis of field infections. On this way, it would be possible to examine large number of serum samples in a short time for the presence of the specific antibodies in large-scaled epidemiological studies. In addition, these proteins that are of great importance as vaccine candidates would be possible to use in the vaccine development studies.

In summary, production of immunoreactive recombinant proteins usable for serological diagnosis of Babesia ovis infection is performed as follows in the light of the above information;

> Immunoscreening of cDNA library constructed from Babesia ovis by using anti-Babesia ovis antibodies,

> Cloning the genes encoding the immunoreactive proteins in the pGEX expression vectors,

> Transformation of recombinant vectors to the competent E. coli cells for expression of recombinant proteins,

> Expression of the recombinant proteins as GST-fusion proteins in competent Esherichia coli cells with IPTG induction,

> Purification of immunoreactive ^' proteins by glutathione affinity chromatography

> Testing the antigenicity of these proteins in ELISA. These recombinant proteins mentioned above may be used as antigen in the configuration of a diagnostic kit for detection of specific antibodies in the sera of sheep naturally infected with Babesia ovis.