PMAP-23-LK-CBM3融合蛋白的原核表达、纯化与鉴定

目的构建抗菌肽-23与3型碳水化合物结合分子（CBM3）融合蛋白的原核表达载体并进行诱导表达、纯化和鉴定。方法PCR扩增PMAP-23-LK-CBM3融合蛋白，产物经酶切、纯化、回收后与经同样双酶切并回收后的pET21a原核表达载体相连接，连接产物转化感受态DH5α菌，经菌落PCR筛选出阳性菌落并提取质粒进行初步酶切鉴定后进一步行双向测序鉴定，插入序列完全正确者命名为pET21a-PMAP-23-LK-CBM3重组载体，并将其转化感受态大肠埃希菌BL21（DE3），加入IPTG诱导PMAP-23-LK-CBM3融合蛋白表达后提取菌体蛋白行亲和层析纯化，并对纯化产物进行SDS-PAGE鉴定和Western Blot鉴定。结果成功构建了pET21a-PMAP-23-LK-CBM3融合重组质粒，成功诱导、纯化并鉴定出PMAP-23-LK-CBM3融合蛋白，分子量与理论值相符。结论成功表达并纯化、鉴定出了PMAP-23-LK-CBM3融合蛋白，为进一步将其应用于抗菌机制和药理实验的研究奠定了基础。

Prokaryotic expression of fusion protein PMAP-23-LK-CBM3

Objective To construct an expression prokaryotic vector for PMAP-23-LK-CBM3 fusion protein and to purify and identify its expression products. Methods The gene encoding PMAP-23- LK-CBM3 protein was amplified by PCR,cloned into the pET21a expressing vector after being enzyme-digested, gel-purified and reclaimed,and the resultant ligation product was transformed into the competent E. coll. DH5 α .The positive colony was screened out by colony PCR and the plasmid was extracted for initial double enzyme digestion analysis and for further bi-directional DNA sequencing. The pET21a- PMAP-23- LK-CBM3 recombinant plasmid was transformed into the competent E.coli BL21 （DE3）. Bacterial lysates prepared from the IPTG-induced cultures were purified by affinity chromatography and identified by SDS-PAGE for its purity and Western Blot for its fidelity. Results The recombinant plasmid pET32a- PMAP-23- LK-CBM3 was successfully constructed,and a fusion protein with the relative molecular weight of 25 KD was successfully induced, purified and identified. Conclusion The PMAP-23- LK-CBM3 fusion protein has been successfully induced,purified and identified, which can be used in further study on its antimicrobial activity and pharmacological effects.