Purification Of The P32.7 Surface Proteins Of N.bombycis And The Analysis Of Relative Field Of P30.4 Gene

Microspora is a large and diverse group of the parasitic protozoa without mitochondria. They are known to cause diseases in fish and insects such as silkworms, bees and locusts. After Nageli found the Aosema bombycis, there are five genus of microsporidia pathogenic from the silkworm having been found in the last two decade. They are Kosema, Pleistophora, Thelohania, Vairimorpha and Endoreticulatus. i\!. bombycis is quarantined in egg production of silkworm because it tends to have offspring infection.This paper studied the total proteins and the surface proteins of N.bombycis and purified the surface proteins and cloned the related gene. l.Separation and Purification of the surface protein P32. 7 of N.boobycisN. bombvcisvas purified on to a discontinuous Percoll gradient. The spore purified is homogeneously, refractive and without other tissue and bacteria shrebs; and can be used in the further experiments.The extracting procedure of protein was based on that of Irby (1986) with modifications described by Guo et al (1995): Spores were suspended in the extraction buffer. The supernatant containing the surface proteins was used in SDS-PAGE.Microscopic examination of the spores after treatment with SDS showed that the shape of spores has few changes. But the spore became sraaller, its ability of movement weakened and its ability of causing diseases of silkworms reduced.The band of P32. 7 of N.bombycis was roughly purified by preparative SDS-PAGE described by Laemmli (1970) . And it was further purified by reversed-phase high pressure liquid chromatography with Spherisorb CB 300A C18column. After the SDS-PAGE analysis, the P32. 7 showed a single band and the HPLC analysis showed a single peak.5The 32. 7kD peptide was transferred onto PVDF membrane after SDS-PAGE, and was seguenced by Bei jin Universitv- The N-terminal amino acid sequence is N-Glu Pro Thr Arg His His Asp Arg Tyr Ala-C. 2.The SDS-PAGE analysis of ali proteins of N.bonbjcisUsing the polar filament inspiring to distill the total proteins of N. bombycis, and analyze it by SDS-PAGE. The result showed that the total proteins had complex band types and including over 30 bands.3. The analysis of 5' flank sequence of the surface protein P30.4The cDNA of the surface protein P30.4 of N.bombycis has been cloned and sequenced in our laboratorv. But after further analysis, there is a lack of start codon in this seguence and it is incomplete in the analysis of the structure and function of this gene.Synthesizing primers by the cDNA sequence of P30.4 and using Rapid Amplification of cDNA Ends (RACE). The RACE PCR was carried out using the reverse transcription product of mRNA of ,V! bombycis as templates. One major band about 400bp was revealed.Sequencing analysis result shows that it has 395 bases, special primers included. The homology between this sequence and the cDNA sequence of P30. 4 is 97% by BLASTN analysis in NCBI.4. The analysis of intron sequence of the surface protein P30.4Synthesizing forward and reverse primers by cDNA sequence obtained and the related PCR was carried out by genome DNA of N. bombycis. Two major bands (Band l is about 400bp and band 2 is 750bp) were obtained. And we cloned the two bands.After cloning and sequencing the two bands, band 2 has 781bp.There are 168bp in band 2 with a homologv of 84% with the Yersinia pestis strain. But the sequence of band l has 360bp and only one primer and there is no homology in NCBI. The structure and function of the two sequences need to be further studied.