Specific Expression Analysis, Preparation Of Polyclonal Antibody Of Serpin6from Bombyx Mori

In order to explore the role of Bmsserpin-6in pigmentation of the epidermis, moltingmetamorphosis and innate immune reactions of Bombyx mori. This investigation employ silkworm(Bombyx mori), strain Da Zao as material, we used real-time fluorescentquantitative PCR method to detect the transcription levels in multiple tissues of differentdevelopmental stages; the transcription levels were also detected in the fat body andhemdympn by induced of lipopolysac charide in the third day of5th instar larvae. E.coliprokaryotic expression system was used for expression study of serpin6gene in vitroalso the fusion protein of serpin6was purified and the purified fusion protein was usedas antigen to immunize KM mouse for the sake of producing polyclonal antibodyagainst serpin-6. The main research consequence are as follows:（一）Specific expression analysis of Bmserpin-6（1）The transcriptional levels were observed in the head, epidermis, fat body,midgut and silk gland of4th instar larvae by the qRT-PCR. But there is highlytranscriptional level in epidermis, There are also higly expression levels in theepidermis the genes of melanin synthesis. so the result inicated Bmserpin-6is related tothe pigmentation of the epidermis. The transcriptional levels were observed duingmolting and metamorphotic process, but there is highly transcriptional levels in moltingthan pre-molting and newly exuviated. The transcriptional levels were significantlyhigher in the newly exuviated. The results show that Bmserpin-6were related to theharding and blackening of epidermis duing molting and metamorphotic.（2）The transcriptional levels of Bmserpin-6in the epidermis were significantlyhigher than other tissues in the second day, sixth day, seventh day of5th instar larvae.But, The transcriptional levels of Bmserpin-6in the head were significantly higher thanother tissues in the first day, third day, fourth day of5th instar larvae. Thetranscriptional levels in the head of5th instar larvae showed a tendency of riseing fromthe first day to the fourth day and then falling suddenly from the fourth day to the seventh day. There were the same trendency with the secretion of juvenile hormone.Silkworm head as the main endocrine organ. The result indicated Bmserpin-6are relatedto the hormone secretion.（3）The expression analysis of Bmserpin-6were induced by lipopolysac charideThe transcriptional levels were increased in the hemdympn and fat body of thethird day of5th instar larvae induced by LPS, The highest transcriptional levels wereobserved in hemdympn at9h after injection, and then gradually redued to the normallevel. The highest transcriptional levels were observed in fat body at6h after injection,but there was a peak at24h.（二）Prokaryotic expression and preparation of polyclonal antibody ofBmserpin-6（1）After being induced by IPTG, we acquire prokaryotic expression recombinantfusion protein which was subsequently purified by Ni column. The molecular weight ofthe purified protein was identified by SDS-PAGE.（2）which was employed as antigen to immunize KM mouse by four separateimmunizations and ultimately achieving polyclonal antibody serum against Serpin6.Theacquired antibody was validated by Elisa and Western blot also its titer was1:20000.In this study, we analysis the transcription of Bmserpin-6by real-timequantitative PCR, the results show that, there are specific transcription in the epidermisand head tissues. Presumably the Bmserpin-6involved in the blackening and hardeningof epidermis, and may be associated with the secretion of hormone in the process ofmetamorphosis. The results show Bmserpin-6mainly involved in the fat body immuneresponse after induced by lipopolysac charide. The study also successfully preparedpolyclonal antibody, lay the foundation for the study of protein distribution.