Cloning,Expression Of ROP2 Of Toxoplasma Gondii And Preparation Of Monoclonal Antibody

Toxoplasma gondii is an obligate intracellular parasitic protozoa,presenting global distributing,the cumulative T.gondii infected persons all over the world reached more than one billion people.toxoplasmosis is a zoonotic parasitic disease which cause a variety of diseases,for instance,toxoplasma encephalitis,eye inflammation,necrosis of pregnant women and abortion of pregnant domestic animal and fetal death.There are three main ways of infection: oral infection,blood infection and placental infection,oral infection has the highest proportion,such as eating raw meat or water containing T.gondii.The only final host of T.gondii are cats,so the pregnant woman should avoid contact with the cat as much as possible.So far,the market temporarily no anti toxoplasma effective vaccine,the most commonly used and effective therapy for patients with toxoplasmosis is pyrimethamine combined with sulfamidics.T.gondii contains four different genotypes: I type,II type,Ⅲ type and atypical genotype,they all have the ability to invade and infect the body,and type II strains of T.gondii infected people that caused the toxoplasmosis is the common.Prugniaud(PRU)of T.gondii strains are type II weak strains which source separated from France,and it has the characteristics of easily invade nerve cells,can invade the central nervous system(Central Nervous,System,CNS)through the blood-brain barrier that infection of various neural cells of the hosts,caused the mechanical damage and dysfunction of the brain,such as learning and memory ability damage.In our research,we used the DNA of the T.gondii PRU strain as template,and ROP2 gene sequence of T.gondii ME49 strain on ToxoDB as the reference sequence,designed specific primers,and obtained ROP2 target genes by PCR amplification.Through sequence alignment,it is found that the target gene of ROP2 obtained from molecular cloning can be used for subsequent protein expression and Monoclonal antibodies(mAbs)preparation.Toxoplasma mAbs have great practical value and they are promising.The research and preparation of Toxoplasma mAbs have a paving effect on the deep study of Toxoplasma gondii.According to the related reports of the anti-Toxoplasma mAbs preparation of at home and abroad,the anti-Toxoplasma mAbs was used to establish the antigen detection method,such as the sandwich ELISA method,etc.Although there are reports that mAbs has a certain resistance to T.gondii infection,there is no definite evidence.We successfully expressed the target ROP2 protein of T.gondii.After purification and renaturation,kunming mice were inoculated with B cells to secrete specific anti-ROP2 antibodies.Finally,the mAbs against ROP2 antigen of the T.gondii was finally prepared by fusion with myeloma cells.It was verified that the antibody could be specific to ROP2 protein,and the antibody was stable without cross reaction.