【摘要】：目的（1）克隆中國(guó)安徽流行區(qū)間日瘧原蟲達(dá)菲血型結(jié)合蛋白II區(qū)基因（Plasmodium vivax Duffy Binding Protein region II，PvDBPII）；（2）體外原核表達(dá)和鑒定重組PvDBPII蛋白。 方法（1）根據(jù)GenBank收錄的間日瘧原蟲Sa1-1株P(guān)vDBPII基因（GI：156081788）序列設(shè)計(jì)引物，從鏡檢陽(yáng)性的間日瘧原蟲感染血樣本中抽提DNA為模板，，應(yīng)用PCR技術(shù)從間日瘧基因組DNA中擴(kuò)增DBPII基因。PvDBPII PCR產(chǎn)物與pET-28a載體分別經(jīng)Nco I和Hind III雙酶切后連接克隆入原核表達(dá)載體pET28a(+)中，構(gòu)建pET28a(+)-PvDBPII重組表達(dá)質(zhì)粒，采用雙酶切及測(cè)序方法鑒定重組質(zhì)粒。（2）將重組質(zhì)粒pET28a(+)-PvDBPII轉(zhuǎn)化至大腸埃希菌BL21(DE3+)中，IPTG誘導(dǎo)表達(dá)帶有His標(biāo)簽的重組蛋白，采用鎳柱親和層析純化重組蛋白，采用SDS-PAGE電泳和Western Blotting分析鑒定表達(dá)產(chǎn)物。 結(jié)果（1）PCR體外擴(kuò)增得到目的基因片段長(zhǎng)約1.1kb。雙酶切及測(cè)序結(jié)果顯示目的基因己被正確地連接入重組質(zhì)粒pET28a(+)中，其插入片段序列與Sa1-1株P(guān)vDBPII基因參考序列相比存在四個(gè)非同義突變位點(diǎn)（R319G、D384G、R390H和L424I）。（2）重組質(zhì)粒轉(zhuǎn)入大腸埃希菌后，誘導(dǎo)表達(dá)的重組蛋白分子量約為44kDa，且能被間日瘧患者血漿特異性識(shí)別。在37℃，IPTG0.5mmol/L誘導(dǎo)表達(dá)4h可達(dá)到最大表達(dá)量，表達(dá)產(chǎn)物為不可溶包涵體。 結(jié)論成功克隆了中國(guó)安徽流行區(qū)間日瘧原蟲PvDBPII基因，表達(dá)出了重組PvDBPII蛋白，并優(yōu)化了PvDBPII重組蛋白的原核表達(dá)條件，驗(yàn)證了其抗原性，為進(jìn)一步研究基于PvDBPII的紅內(nèi)期間日瘧疫苗提供了基礎(chǔ)；
[Abstract]:Objective (1) to clone the gene (Plasmodium vivax Duffy Binding Protein region II,PvDBPII); (2 of II region of Tamiflu blood group binding protein of Plasmodium vivax in Anhui Province, China, and to express and identify the recombinant PvDBPII protein in vitro. Methods (1) primers were designed according to the PvDBPII gene (GI:156081788) sequence of Plasmodium vivax Sa1-1 strain included in GenBank, and DNA was extracted from infected blood samples of Plasmodium vivax positive by microscope as template. The DBPII gene, PvDBPII PCR product and pET-28a vector were amplified from vivax malaria genomic DNA by PCR technique and cloned into the prokaryotic expression vector pET28a () by double enzyme digestion of Nco I and Hind III, respectively. The recombinant expression plasmid of pET28a () -PvDBPII was constructed. The recombinant plasmid was identified by double enzyme digestion and sequencing. (2) the recombinant plasmid pET28a () -PvDBPII was transformed into Escherichia coli BL21 (DE3) to induce the expression of recombinant protein with His label, and the recombinant protein was purified by nickel column affinity chromatography. The expressed products were identified by SDS-PAGE electrophoresis and Western Blotting analysis. Results (1) the length of the target gene fragment was about 1.1 kb by PCR amplification in vitro. The results of double enzyme digestion and sequencing showed that the target gene had been correctly ligated into the recombinant plasmid pET28a (). Compared with the reference sequence of the PvDBPII gene of Sa1-1 strain, there were four non-synonymous mutational sites (R319GN, D384GN, R390H and L424I). (2) in the recombinant plasmid transformed into Escherichia coli. The molecular weight of the recombinant protein induced was about 44 kDa and could be specifically recognized by the plasma of vivax malaria patients. The expression of IPTG 0.5 mmol / L at 37 鈩

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