Epidemiological Investigation On Laboratory Rats And Mice Helicobacter And Expression Of Helicobacter Hepaticus FlaB Gene In E.Coli

Rodent Helicobacter, a member of the family Helicobacter, are gram-negative curved to spiral shaped bacteria that prevalently colonizes digestive tract of rodent animals by ways of subclinical in infection, such as rats, mice, gerbils, and so on. However, some strains of Helicobacter species can result in various kinds of inflammations, such as hepatobiliary inflammation, typhlitis, colonitis and even malignant tumors in immumodeficient rodents. H. hepaticus is the most representative of rodent helicobacter in inducing diseases. It is another well-defined carcinogen of helicobacter. Infected with helicobacter, the parameters of immune response in vivo will be changed and then the results of experiments tend to be interfered. It has been numerous reports on infection of rat and mouse colonies with Helicobacter have become an increasing concern for the research community in abroad. Thus it is necessary to establish a quick and accurate diagnostic method.Rodent Helicobacter has been classified as a kind of pathogen by most countries and ICLAS which has to be eliminated on rodent laboratory animals. However, it has not been listed into our country’s Government Standard owing to absence of one effective detecting method and standard strains. In this experiment we isolated, cultivated and identified the helicobacter isolates. To provide reference and basis for the establishment of Chinese laboratory animal grades and monitoring standards, we identify the prevalence of rodent helicobacter infection in laboratory rats and mice. Hh-flaB is expressed by prokaryotic expression system pET-flaB, and the expressed rflaB can be a candidate antigen for the establishment of new ELISA method.1. Isolation, cultural and identification of Helicobacter hepaticus:Columbia selective agar supplemented was used to isolate H. hepaticus from the ileocecal junction of immunosuppressive mouse under standard micro-aerobic conditions at37℃. After amplification, suspected bacterial colony was identified by Gram’s staining, biochemical characterization and gene sequencing by amplifying16srRNA gene. The16srRNA sequences of the helicobacter isolated has98%similarity to the standard strain ATCC51449. The helicobacter strains isolated from C57BL/6mouse is ascertained H. hepaticus.2. Epidemiological investigation on laboratory rats and mice Helicobacter in the neighborhood of Shanghai. PCR detecting percentage in laboratory rats and mice as follows:the positive rate of rats was70.3%(71/101), among them, the positive rates of clean grade and SPF grade were69.6%(48/69) and71.9%(23/32) respectively; the positive rate of mice was35.8%(126/352), among them, the rates of clean grade and SPF grade were51.5%(52/101) and29.5%(74/251) respectively. ELISA detecting percentage in laboratory rats and mice as follows:the positive rate of rats was52.7%(87/165), among them, the positive rates of clean grade and SPF grade were53.6%(45/84) and51.9%(42/81) respectively; the positive rate of mice was15.9%(14/88), among them, the positive rates of clean grade and SPF grade were19.2%(5/26) and14.5%(9/62) respectively.Further analysis the infection types of the rodent helicobacter in rats and mice by PCR amplification five kinds of rodent helicobacter16SrRNA gene, the results showed Helicobacter species infection were be exist in laboratory rats and mice of the neighborhood of shanghai. H. rodentium, H. hepaticus and H. bilis were the most common Helicobacter species in rats and mice.3. Expression and purtification of Helicobacter hepaticus flaB gene and preparation of antibodies against flaB. The DNA coding sequence for Hh-flaB was synthesized and amplified by PCR, the resulting PCR product was cloned into pGEM-T vector. After identification by sequencing analysis, flaB gene was subcloned into prokaryotic expression vector pET-32a. The confirmed recombinant clone pET-flaB was transformed into E.coli BL21(DE3) and induced to express proteins by IPTG. The expression of flaB fusion protein was identification by SDS-PAGE and Western blot. The fusion protein, approximate54kDa, was purified through6×His binding. To obtain the antibodies against flaB, the purified fusion protein was immunized the BALB/C mouse. Determine flaB fusion protein immunoreactivity and antigenicity by Western blot using mouse anti-Hh antibody and by indirect ELISA using mouse anti-flaB serum respectively. The flaB fusion protein was recognized by the antibodies against whole cell of H.hepaticus. It also induced high titer serum antibody in mouse. A prokaryotic expression system for high expression of Hh flaB was constructed, and the expressed the recombinant protein flaB showed good immunoreactivity and antigenicity.