Study On Cloning And Expression Of HSP20 Gene Of Babesia Bigemina And Immunorectivity Of Its Recombinant Protein

The Babesia bigemina is a kind of very serious blood protozoiasis. Its mainly clinically character is fever, anaemia,hemoglobinuric and death[1]. Boophilus microplus is one of the important transmitters. This disease would be not killed thoroughly when it diffuse to an area. Because the Babesia bigemina can be delivered through placenta[2]. So the pasture that has been infected Babesia bigemina can not become safty pasture in very short time. The Babesia bigemina does harm to the cattle industry. Nowadays, microscope observation method is still the mainly method in detection in basement veterinarian of our country. The necessary terms that definite this disease is finding protozoon in blood film[3,4]. But the shape of Babesia bigemina is changing follow the course of disease. It takes some difficult for detection. So it is very necessary to establish a blood serum detection system that is very convenience, accurate and cheap to final diagnosis this diease quikly and timely. The following three studies were conducted in the present article.1 The Babesia bigemina HSP20 gene cloning.The Babesia bigemina HSP20 gene has 699bp basic radical. It has a intron. This gene can be translate into a 20KDa protein. Cloning this gene by PCR through the whole blood genome, making a vector, then identificate it by sequencing. The result show that the cloned Babesia bigemina HSP20 gene has four mutation. The mutation in 120bp (A to G) and 191bp (T to C) in intron. they do not cause changing of protein. The mutation in 582bp (G to C) is a samesense mutation. Only the 643bp mutation (A to G) change the code of protein—THR to Val.2 Prokaryotic expression of Babesia bigemina HSP20 gene, purification of recombinant protein and its immunoreactivity study.The total RNA was extracted from whole blood of infected cattles. The exon of Babesia bigemina HSP20 gene was amplified by RT-PCR. It was put into Prokaryotic expression plasmid—pGEX-4T-HSP20(exon) and was expressed in E.coli BL21(DE3).3 Eucaryotic transient expression of Babesia bigemina HSP20 gene, purification of recombinant plasmid and its immunoreactivity study.The cloned HSP20 gene was recombined into Eucaryotic expression plasmid—pcDNA3.1+. The Eucaryotic expression plasmid—pcDNA3.1(+)-HSP20 was established. After identifuication by sequencing, inject it into vena caudalis of mouse to express transiently of Babesia bigemina HSP20 gene. Extract the total RNA of delivery, a stripe of DNA in 534bp can be found by RT-PCR. That means the success of Eucaryotic transient expression. The sensitive counteractive antibody of this plasmid can be aroused in immuned mouse.Babesia bigemina HSP20 gene was cloned and expressed very successful in this study. The recombined protein that was purified has strong bioactive. It is the basement of establishing the ELISA detection system.