Studies On The Muscle Larval Antigens Of Trichinella Spiralis And T. Pseudospiralis

The genus Trichinella is presently divided into 8 species(Trichinella spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae and T. zimbabwensis) and 3 additional genotypes, Trichinella T6, T8 and T9, whose classifying status are not determined. T. pseudospiralis, T. papuae and T. zimbabwensis are non-encapsulated species. Because of distinct differences of different Trichinella species, such as, geography distribution, the range of host, the infecting ability to host, immunity response of host to different species, it is very important for pathogenesis, immunodiagnosis and immunoprophylaxis to study antigens of Trichinella.To observe difference of antigens of muscle larvae of Trichinella spiralis and T. pseudospiralis, this study analyses soluble antigens and excretory-secretory(ES) antigens of muscle larvae of Trichinella spiralis and T. pseudospiralis with using sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and two-dimensional electrophoresis(2-DE) techniques.Materials and Methods1. Trichinella species and experimental animals Trichinella spiralis (T1) and T. pseudospiralis (T4) obtained from International Trichinella Reference Centre were maintained and passaged in Kunming mice. 4 weeks-old specific pathogen-free SD rats were obtained from the Experimental Animal Center of Medical College of Zhengzhou University. The rats infected with different species of Trichinella were killed in 8 weeks after infection, the carcasses were digested and purified muscle larvae were collected.2. Preparation of soluble and ES antigen of muscle larvae Muscle larvae were mixed with lysis buffer and homogenized on ice by strokes with a Teflon pestle in a glass tissue grinder about 1～2 hours. After this, the mixture was further homogenized with ultraso-nication. The lysis solution was clarified by Centrifugation and collected. The sterilized muscle larvae were cultured in serum-free RPMI-1640 medium at 37℃, 5% CO₂ for 20～24 hours. The culture supernants were collected, centrifugated, dialysed, lyophilized and muscle larvae ES antigens were obtained. Soluble antigens and ES antigens of muscle larvae were measured with Bradford protein assay reagent, subpackaged and stored at -80℃.3. SDS-PAGE It was analyzed by the SDS-PAGE that soluble antigens and excretory-secretory(ES) antigens of muscle larvae of T. spiralis and T. pseudospiralis. The gel was stained and bleached sufficiently, molecular weight bands were revealed, and then cameraed, analyzed by Gene Genius gel image analysis system. Different protein bands of T1 and T4 were discriminated.4. 2-DE To obtain 2-DE profiles, the soluble proteins were firstly separated by Isoelectric Focusing(IEF) electrophoresis and separated according to difference in molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the second dimension. Repeat for 3 times. The gels were stained, then were scanned with ImageScanner (PowerLook 1120, 300bpi and 8bits) to get the images. Image analyses were conducted with Image Master 2D Platinum 6.0.Results1. SDS-PAGE profiles of soluble antigens of T1 and T4 muscle larvae Analyzed by SDS-PAGE, the soluble antigens of T1 muscle larvae show 22 bands(221.62～14.88 kDa), among which the protein amount of 5 major bands(115.19, 65.56, 53.93, 29.08, 14.88 kDa) accounting for 47.4% of total soluble proteins; T4 muscle larvae shows 18 bands(185.28～14.27 kDa), 7 major bands(119.30, 68.56, 56.1, 48.50, 40.74, 28.36, 14.27 kDa) accounting for 66.7%. 6 bands(59.72, 44.37, 23.66, 22.36, 18.26, 16.34 kDa) are T1 specific in soluble antigens, and 4 bands(132.60,119.30, 35.26, 31.02 kDa) are T4 specific.2. SDS-PAGE profiles of ES antigens of T1 and T4 muscle larvae The ES antigens of T1 muscle larvae represent 10 bands(113.21～14.37 kDa), within them 4 major proteins (55.65, 50.41, 43.61,14.37 kDa) amount accounting for 85.7%; T4 muscle larvae represent 9 bands(104.71～14.51 kDa) and 4 major proteins amounts account for 81.3%.The patterns of ES antigens of T1 and T4 muscle larvae are different.3. Comparisons of soluble and ES antigens SDS-PAGE profiles of T1 and T4 muscle larvae In T1 muscle larvae proteins, molecular weights of 8 T1 soluble antigens (115.19, 53.94, 48.97, 44.37, 29.08, 22.36, 19.27, 14.88 kDa) are similar with 8 ES antigens (113.21, 55.65, 50.41, 43.61, 31.42, 22.21,19.67, 14.37 kDa). For T4, molecular weights of 5 soluble antigens(56.11, 35.26, 28.36, 20.65, 14.27 kDa) are similar with ES antigens(54.58, 36.80, 28.91, 21.70,14.51 kDa). 4. 2-DE of soluble antigens of T1 and T4 muscle larvae The soluble antigens of T1 muscle larvae represent 193±12 protein points mainly in the molecular weight areas of 11～22 kDa,25～64 kDa and 100～144 kDa, respectively pI(isoelectric point) is 4.7～8.2, 4.5～6.5 and 5～7, with 150 matching points and matching ratio is 81%; T4 muscle larvae represent 175±9 protein points mainly in the molecular weight areas of 12～21 kDa and 25～90 kDa, respectively pI(isoelectric point) is 4～9.5 and 4.5～9.6, with 141 matching points and matching ratio is 82%5. 2-DE of ES antigens of T1 and T4 muscle larvae The ES antigens of T1 muscle larvae represent 82±6 protein points mainly in the molecular weight areas of 13～16 kDa, 18～22 kDa and 40～55 kDa, respectively pI(isoelectric point) is 4～7,3.8～6.2 and 5～9, with 74 matching points and matching ratio is 84%; The ES antigens T4 muscle larvae represent 69±5 protein points mainly in the molecular weight areas of 10～15 kDa, 17～25 kDa and 29～55 kDa, respectively pI(isoelectric point) is 4.7～6.5, 4.6～6 and 5～7, with 57 matching points and matching ratio is 80%.Conclusions1. The protein components in muscle larval soluble antigens of T. spiralis and T. pseudospiralis are more than that in their ES antigens.2. The protein components in muscle larval soluble and ES antigens T. spiralis are different from that T. pseudospiralis. Both of the soluble and ES antigens of T. spiralis muscle larvae are more complex than that of T. pseudospiralis.