Qingdao Amphioxus Body Surface Mucus Lectin Nature And Masp Gene Cloning

In this paper, the presence of lectins in the skin mucus of amphioxus was verified by the aggutination assay and the effects of physical and chemical treatments on hemagglutination activity of amphioxus skin mucus were tested by the means of heat treatment and pH treatment. The skin mucus showed agglutinating activity against all human A, B and O, rabbit, chick, grass carp and toad erythrocytes tested. Rabbit erythrocytes, however, invariably possessed the highest titer among the cells derived from different animals including human. In addition, there is no blood-group specificity toward human erythrocytes. The hemagglutinating activity of amphioxus skin mucus was decreased after heating at 60 ℃ for 30 min, and was almost lost upon heating at 100℃ for 30 min. the hemagglutinating activity of the skin mucus remained stable in the pH range from 6 to 10, but it was inactivated at pH above 10 or below 6. The hemagglutinating activity in the skin mucus is inhibited by D-galactose, which is same with the hemagglutinating activity of the humoral fluids of amphioxus. These results indicated that lectins present in the skin mucus of amphioxus and defend amphioxus from the invasion of pathogene.The MASP ( mannose-binding lectin-associated serine protease ) gene has been cloned by the method of degenerative PCR and the fragment of the PCR product is 630 bps in length. Sequence alignment shows that the fragment of PCR product showed some identities to the MASP gene of Xenopus laevis, Branchiostoma belcheri , the trypsin gene in Litopenaeus vannamei and the serine protease gene in Aurelia aurita. Considering of the specificity of the degenerative primer designed in this PCR reaction, the identity between the sequence we wanted and the fragment of PCR product and the presence of AsMASPa, AsMASPb, Clr and Cls ( the homologous gene of MASP gene ) in Halocynthia roretzi, a Japanese ascidian, we believe that the sequence of PCR product is some part of the MASP gene or MASP homologous gene. All these results suggest that there is some simple immunological reaction including lectins in amphioxus and some deduced or absent complement pathway such as lectin pathway which was activated by the complex of MBL-MASP.Although there are some reports about MASP or MASP homologous genes in human being, rat, xenopus, shark, lamprey and ascidian, there is no amphioxus MASP homologus gene reported in amphioxus. Amphioxus, an existing animal that belongs to the phylum Chordata, subphyla Cephalochordata, is always regarded as the invertebrates which is the nearest to the vertebrates. It has great sense on the research of how the vertebrates develop from invertebrates. So, the cloning of amphioxus MASP gene is very important in the development of MASP gene and the immune system, even the origin of the vertebrates.