摘要
利用基因工程技术制备抗原性好的弓形虫GRA6蛋白和P30蛋白的融合蛋白 ,并用作抗原检测弓形虫抗体。根据弓形虫GRA6蛋白和P30蛋白的氨基酸序列 ,通过计算机分析 ,筛选出其中较强的抗原决定簇。用PCR方法分别扩增含抗原决定簇的基因片段。将这两个基因片段克隆至同一质粒pET2 8a(+)内 ,表达一个融合蛋白。将重组质粒转化大肠杆菌BL2 1(DE3) ,筛选表达该融合蛋白的工程菌。纯化表达的融合蛋白 ,用已知的 6份抗弓形虫IgM阳性血清和大量正常人血清 ,ELISA法检测纯化融合蛋白的抗原性和特异性。获得了高效表达含弓形虫GRA6蛋白和P30蛋白抗原表位的工程菌 ,表达的融合蛋白约占菌体蛋白总量的 2 5 %。纯化获得了表达的融合蛋白 ,该蛋白有较好的抗原性和特异性。表达的弓形虫GRA6和P30融合蛋白可用做抗原检测弓形虫抗体 ,用于临床及孕妇检测 ,对优生优育有较大意义。
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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第6期674-679,共6页
Chinese Journal of Biotechnology
关键词
弓形虫
GRA6
P30
融合蛋白
基因表达
Toxoplasma gondii, p30, GRA6, expression and purification, ELISA
