Prokaryotic Expression And Immunogenicity Of Recombinant AnkA Protein Of3Isolates Anaplasma Phagocytophilum

Anaplasma phagocytophilum is a0.2-1.0um Gram-negative bactria,which is a tick-borne and strictly intracellular parasitic, to mainly cause human granulocytic anaplasmosis (HGA). Since nineteen ninties it was found in the United States for the first time, European, Australia, South Korea and China has had reported, and the upward trend in recent years. In2006, Anhui Wuhu yijishan hospital HGA nosocomial infection event was China’s first reported confirmed infection case, and also the world’s first report of human granulocytic anaplasmosis human-to-human transmission event. Because the disease clinical manifestations were acute fever, muscle pain, fatigue, headache and other symptoms, which was similar to virus infection. The disease was lacked clinical awareness, no better rapid diagnostic method, so the disease was easy misdiagnosis, seriously can cause multiple organ failure, and even death.In order to explore different human isolates of AnkA protein immunogenicity are different or not, to further reveal the possible structure and immune epitope differences and possible Protein Profiling differences associated with the host cell. The first use of the DNAMAN software, the United States Webster strain, Slovenia1567strain and the United States96HE58strains, which three AnkA proteins amino acid sequences alignments, the consistency of three strains of AnkA protein was90.2%, the consistency of Webster strain and96HE58strain AnkA protein was the highest, is95.2%,1567strain and96HE58strain AnkA protein matched minimum, is90.6%. B lymphocyte linear epitope prediction of AnkA protein of3isolates of A.phagocytophilum by Protean indicated that Webster strain and96HE58strain isolates epitope of AnkA protein there is little difference, and Webster strain and96HE58strain with1567strains of AnkA protein antigen epitope prediction results more differences.We formed3strains human isolates ankA gene by prokaryotic expression, and made preliminary analysis immunogenicity. Anaplasma phagocytophilum Webster strains, Slovenia1567strains, the United States96HE58strain ankA gene sequences amplified by PCR, constructed pET-30a-ankA expression vector, transformed into Escherichia coli BL21, IPTG induced fusion protein high expression, the amino acid sequences were identification by flight mass spectrometry. preparing polyclonal antibody by immune BALB/c mouse. Using ELISA and Western blot method to analyze immune response mutually and cross reactivity of three strains’s AnkA recombinant protein interact with sera antibodies immunized mice.Simultaneous determination the reaction of three isolated strains’s recombinant AnkA protein with serum antibody of HGA patients in china.The results showed that the recombinant proteins existed mainly in soluble, immune mouse to obtain high titer anti-AnkA IgG antibody, immune cross between three strains of recombinant protein antibody. Immune reaction presented of three strains of recombinant protein with China Intangible body antibodies.This study successfully prokaryotic expression and purification three strains Anaplasma phagocytophilum AnkA protein, recombinant protein with positive patient serum in our country presented immune response. Three strains AnkA recombinant protein with immune mouse polyclonal antibody cross-reactivity. By Western blot and ELISA method not to get differences in immunogenicity, different human isolates of AnkA protein is not differences significantly.