家蚕丝氨酸蛋白酶bmsp82的研究

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1、摘要细胞外基质的形成，从而保护丝腺细胞免受吐丝过程中丝蛋白对丝腺腔内侧细胞的压力和剪切力损伤。4、家蚕丝氨酸蛋白酶BmSP82的表达及活性初探。通过原核表达获得了带GST标签的BmSP82成熟体，利用GST标签进行了亲和层析，得到了纯度较高的GST-BmSP82重组蛋白。利用TEV酶切除BmSP82重组蛋白的GST标签后与酪蛋白孵育，发现原核表达的BmSP82蛋白没有活性。于是又进行了毕赤酵母真核表达。将丝氨酸蛋白酶基因BmSP82的成熟体片段构建到真核表达载体pPIC9K上，通过电转化转入毕赤酵母GSl15中进行培养。最

2、后运用BMGY和BMMY液体培养基体系进行诱导表达。检测到酵母转化子在诱导24h后显著表达，于是进行了硫酸铵梯度沉淀，得到了较纯的BmSP82的成熟体蛋白，为进一步的蛋白酶活性检测与功能研究奠定了基础。关键词：家蚕，丝氨酸蛋白酶，CUB结构域，表达III西南大学硕士学位论文ABSTRACTResearchserineoroteaseBmSPResearch0nserlneroteaseIt"82ol“苓ZsilkwormBiochemistryandMolecularBiologymastercandidate：YongS

3、upervisor：Prof．PingZhaoSupervlsor：I-'rotlng11aoSerineproteasesareenzymesthathydrolyzeproteins，namedfromtheSerattheactivesitewhichshownucleophilicattackonsubstrate．Asanimportantmemberoftheproteasefamily，theirroleistobreakthepeptidebondsofproteinmolecules，andmakethe

4、mbecomesmallproteins．Thecatalysisisachievedbytheconformationalchangeofaminoacidresiduesattheactivecenter．Theremustbeaserineattheactivecenterofprotease，SOwecallitsefineprotease．Inmammals，serineproteasesplayveryimportantroles，manyproteinsarewell—knownintheserineprot

5、easesfamily，includingdigestiveenzymes，clottingfactorsandmembersofthecomplementsystem．Becausethatserineproteasesplayimportantrolesinembryonicdevelopment，tissueremodeling，celldifferentiation，angiogenesisanddefenseagainstpathogen，therearelotsofresearchreportsonanimal

6、serineprotease．However，studiesonserineproteasesofinsectarerelativelylittle．Rosseta1．(2003)identifiedtheserineproteasegenefamilyofthedrosophilabasedonthegenomedata．Atotalof19geneswereidentified，beingdividedintoSPandSPHcategorieswhichcontain86and33genes，respectively

7、．Theyevolutionaryanalysisrevealedthatgeneduplicationandmutationplayimportantrolesintheformationofthisgenefamily．Silkwormhasasmanyserineproteasegenes．AvarietyofserineproteasehavebeenfoundindifferenttissuesofBombyxmori．Byreferringtotheserineproteasesequencesinothers

8、pecies，PingZhaoeta1．(2010)comprehensivelyanalyzedtheserineproteasegeneofB．moribaseonthegenomedata．Atotalof143serineproteasegenewereidentifiedfromsilkwor