[Expression, purification and serological reactivity of a chimeric antigen of GRA6 with P30 from Toxoplasma gondii].
Major surface protein (p30) and Dense Granule Antigen GRA6 of Toxoplasma gondii have good antigenicity, and could be used for detection of IgM against Toxoplasma gondii. GRA6 may complement P30 to reach more high sensitivity for detection of antibodies to Toxoplasma gondii, so, we try to express the chimeric protein of GRA6 and P30 by genetic engineering, identify its antignenicity and use for developing diagnosis reagent. Antigenic domains of p30 and GRA6 of Toxoplasma gondii were screened by analyzing their sequences using the software ANTHEWIN. Two DNA fragments encoding respectively antigenic domains of p30 and GRA6 were cloned, they were inserted into the same expression vector pET28a( + ) and expressed as a chimeric protein in Escherichia coli. BL21(DE3), the expressed chimeric protein of p30 with GRA6 in a form of inclusion body was about 25% of total proteins of E. coli. BL21(DE3). The inclusion body was washed once with 0.5% Triton X-100 and dissolved with 0.5% SKL, after renaturation by gradient dialysis, the recombinant protein was purified by DEAE-Sepharose FF cation column and then detected with 12% SDS-PAGE, it exists mainly in the eluted peak with 300 mmol/L NaCl and has high purity. By using enzyme-linked immunosorbent assay (ELISA), the recombinant protein was examined for reactivity with immunoglobulin M (IgM) antibodies in 6 sera from patients infected with Toxoplasma gondii ., it was reactive with all the 6 sera but not with sera from normal people, these results showed that the recombinant chimeric antigen has good antigenicity and specificity and could be used for detection of IgM against Toxoplasma gondii. The expressed chimeric protein could be used for epidemic investigation of Toxoplasma gondii, blood donor screening, especially for detection of pregnant women, and is of great significance in prevention of Toxoplasma gondii infection.
Li
Yue-Xi
YX
East-China Institute of Medical Biotechniques, Nanjing 210002. liyxi@163.net
Zhang
Jin-Hai
JH
Tao
Kai-Hua
KH
Huang
Pei-Tang
PT
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology*
D000953:Antigens, Protozoan; C095047:GRA6 protein, Toxoplasma gondii; D007075:Immunoglobulin M; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C047168:SAG1 antigen, Toxoplasma