Expression And Application Of Recombinant Genes SAG2, GRA6, GRA7 Of Toxoplasma Gondii

Background and objective Toxoplasma gondii is a serious opportunistic protozoa which effected people’s health. It can spread between humans and livestock around the world. Toxoplasma gondii can violate all tissue’s nucleated cells except red blood cells. People with normal immunity are in asymptomatic Plasmodium state after Toxoplasma gondii infection. But for the special crowd suffering from AIDS, tuberculosis, cancer, organ transplantation, whose immune function is damaged, infection can result in obvious clinical symptoms, or even death. Toxoplasma gondii infection in pregnant woman can cause fetal malformation, abortion and stillborn foetus. Spreading in poultry leads to poultry deaths and serious economic losses. So, the prevention of toxoplasmosis has great clinical value in the early diagnosis of toxoplasmosis. Clinical diagnosis is very difficult because Toxoplasma parasite on host cells, it is difficult to get directly. Clinical manifestations are not special. The most commonly used methods are bacteriology, immunology and molecular biology, each method has advantages and disadvantages. Microscopy directly is a traditional method, it is of high specificity but easily missed, which can not meet the clinical needs. Immunological diagnostic method is of high sensitivity and specificity, relatively convenient, so the application of immunological diagnosis of toxoplasmosis is more important. With the development of molecular biology in recent years, the diagnostic value of Toxoplasma antigen molecule cloned into prokaryotic or eukaryotic expression vector for expression and purification, to obtain high-purity antigen specificity, are widely used. SAG2 is the surface antigen, and GRA6 and GRA7 are dense granule proteins, which have strong antigenicity and immunogenicity. In this study; the recombinant plasmids pGEX-4T-SAG2, pGEX-4T-GRA6 and pGEX-4T-GRA7 of Toxoplasma gondii RH strain SAG2, GRA6 and GRA7 gene were constructed, respectively. Then, the recombinant plasmids were transformed into E. coli BL21, and induced with LPTG to express the target protein. Using ELISA to analyze the human serum after the protein was purified. It is pave the way to explore diagnosis kit using SAG2, GRA6 and GRA7 as a human, animal studies toxoplasmosis diagnosis antigen.Methods 1. PCR amplification. Extract genomic DNA RH strain of Toxoplasma gondii from infection mouse ascites, and using it as template for PCR amplification, and then electrophoresis of PCR products were purified.2. Construction of recombinant plasmids. The PCR product and the vector pGEX-4T were double digestion (BamHl, EcoRl), recovered by recycling kit fragments, fragment with the carrier to get the connection, the ligation product was transformed into E.coli TOP10 competent cells. The positive plasmids were sequenced after PCR and restriction enzyme digestion.3. Expression and purification of recombinant SAG2, GRA6 and GRA7. The positive recombinant plasmid were expression by IPTG induced and using SDS-PAGE and Western blot analyze the fusion protein after His-bindTM column purified.Results l.PCR amplification of specific target genes (SAG2, GRA6 and GRA7) identified by electrophoresis PCR products match the length of the theoretical value of about 561,693 and 714bp.2. Recombinant plasmids were digested by BamHI and EcoRI, electrophoresis display appears bands with the expected size, which is consistent with the corresponding position. Recombinant plasmids were sent to be sequenced. The sequencing results are consistent with the original sequence, indicating the recombinant plasmids were constructed successfully.3. Taking samples in each group by SDS-PAGE analysis, the electrophoretic display significant bands with 47KD,52KD and 53KD, indicates that the fusion protein was expressed successfully.4. Using purified protein as coating antigen to analyze 358 samples of human serum Toxoplasma IgG antibody, the results showed that positive rate was 3.07%-4.75%. Then, we used Toxoplasma imported diagnostic kits to detect positive samples and negative samples (48), the results were consistent with the ELISA results.