Purification Of HMWP-B And Homogenous Identification Of HMWP In Sow Milk

1 Research of Purification of HMWP-B in Sow MilkThe sow milk which only have HMWP-B was changed to skim milk after removal of the cream by centrifugation. The skim milk was adjusted to pH4.6 to precipitate most of casein and centrifuged to collect whey. Whey protein was fractionated by different saturation of ammonium sulfate. The precipitation of 40% ammonium sulfate was collected, then dissolved in distilled water and dialyzed to obtain crude sample. The purification of crude sample was researched by isoelectrofocusing preparation electrophoresis, SephadexG-100 column chromatography, DEAE-52 anion exchange chromatography and High Q column anion exchange chromatography. HMWP-B and P -Lactoglobulin can't be isolated by isoelectrofocusing preparation electrophoresis, but can be easily isolated by SephadexG-100 column chromatography. SephadexG-100 column chromatography can't completely isolate HMWP-B and the big molecule such as immunoglobulin, lactoferrin and so on. The chromatography behavior of DEAE-52 anion exchange chromatography and High Q column anion exchange chromatography is similar. They can't purify HMWP-B in sow milk.2 Purification of HMWP-B in Sow MilkHMWP-B crude sample was semi-purificated by SephadexG-200 column chromatography, further purificated by High Q column anion exchange chromatography. Finally , the pure HMWP-B was gained. The purified HMWP-B exhibited a single band on SDS-PAGE by coomassie blue stained. The purity is 92.3%.3 Preparation of Rabbit anti- HMWP-B Polyclonal Antibody and Homogenous Identification of HMWP in Sow MilkHigh pure HMWP-B was prepared by cutting and extracting gel. The polyclonal antibody was prepared by immunizing New Zealand rabbit with the pure HMWP-B. The antiserum liter was 1:4 and 1:1600 by double immunodifusion test and ELISA respectively. The polyclonal antibody has no immunocross reaction with the main protein (casein) insow milk by ELISA. The speciality of the antisemm is good by Western Blot test.From comparing sow milk with cattle and rabbit by SDS-PAGE and Western Blot, HMWP-B might exit only in sow milk. The origin of HMWP-B wasn't blood by Western Blot. So, HMWP-B was deduced to be synthesized and secreted by mammary per se.Western Blot identification of HMWP in sow milk show the A and C protein of HMWP have immunocross reaction with HMWP-B. The different protein of HMWP have same antigen cluster and the homogen of HMWP is very high. So, the hypothesis of the polymorphism of HMWP is true.