Functional Study Of N And N+C Terminals Of Circumsporozoite Protein From P. Berghei Targeting To Hepatocyte

Circumsporozoite protein (CSP) is the major surface protein of Plasmodiumsporozoites and forms a dense coat on the parasite’s surface. CSP can be divided intothree parts roughly; the amino terminal containing region I and signal sequence, acentral repeat region, the carboxyl terminal containing region II andGlycosylphosphatidylinositol (GPI)-anchor sequence. The central repeat region isspecies-specific and immunodominant, containing B cell epitopes. At present, CSPhas emerged as one of the prime candidates for an anti-malarial vaccine anddiagnostic antigen.When the infected Anopheles takes a blood from intermediate hosts (such ashuman, mammals), sporozoites from the mosquito’s salivary glands are injected intothe host’s subcutaneous capillaries and circulate in the peripheral blood. Within30minutes they attach to and invade hepatocytes. The speed and specificity of sporozoitelocalization to hepatocytes suggest a receptor-mediated event. Previous studies haveshown that CSP binds to heparan sulfate proteoglycans (HSPGs) on the hepatocytesurface. GAG is the binding site. In Plasmodium falciparum, for example, thefragments of CSP N terminal (aa82-aa100）and C terminal (aa328-aa346) could bindto HSPGs of hepatocyte specifically. Therefore, CSP has a potential value as ahepatocyte-targeting molecule. As an antigen with strong immunogenicity, CSP could induce specific antibodies to inhibit CSP’s binding to hepatocytes. Taken together, theway to retain its ability tageting to hepatocyte and to reduce its immunogenicity,makes it possible to be a hepatocyte-targeting molecule.ObjectivesCircumsporozoite protein (CSP), a predominant surface antigen on sporozoitesurface, has been associated with binding and invasion of hepatocytes by thesporozoites. We investigated the role of terminals of N and N+C of CSP fromPlasmodium berghei in targeting to hepatocyte. pET28a-PbCSP-N andpET32a-PbCSP-N+C were constructed and the two proteins were recombinantlyexpressed and purified. The recombinant proteins were evaluated for their capacity oftargeting to hepatocyte from normal mouse in vitro and in vivo, respectively.Methods1. Cloning, expression, purification of PbCSP-N and PbCSP-N+CN-terminal and N+C terminal were cloned into the prokaryotic expressionvectors to construct pET28a-PbCSP-N and pET32a-PbCSP-N+C, respectively. Thetwo recombinant proteins were expressed induced by IPTG in E.coli BL21. Then thefusion proteins with6×his tag were purified by affinity column chromatography, andanalyzed by SDS-PAGE and Western-blot.2. In vitro, the recombinan proteins of PbCSP-N and PbCSP-N+C could bind tohepatocytes from normal mouse specificallyThe ability of PbCSP-N and PbCSP-N+C recombinant proteins binding toHSPGs on the surface of hepatocytes from normal mouse were identified bypull-down assay. Then the frozen sections of liver, lung, heart, spleen, kidney,stomach, small intestine, uterus, mesenteric lymph gland from normal mice weremade. The ability of PbCSP-N, PbCSP-N+C recombinant proteins binding tohepatocytes was evaluated by immunohistochemical method.3. In vivo, the recombinan proteins of PbCSP-N and PbCSP-N+C could target toliver from normal miceThe recombinan proteins of PbCSP-N, PbCSP-N+C and Trx protein werelabelled by Na125I. The purified labelled products were injected into normal micethrough tail vein. At1h,2h,4h,6h,12h after injection, the dynamic distribution ofrecombinant protein PbCSP-N, PbCSP-N+C and Trx protein in mouse’s liver, heart, lung, kidney, spleen, small intestine, uterus, urine and blood were detected.Results1. Cloning, expression, purification of PbCSP-N and PbCSP-N+CpET28a-PbCSP-N and pET32a-PbCSP-N+C prokaryotic expression vectorswere constructed sucessfully. The recombinant proteins of PbCSP-N and PbCSP-N+Cwere expressed as a fusion protein with a6×His tag in E. coli upon induced by IPTG.The fusion protein were purified by affinity column chromatography and showed asingle band in SDS-PAGE of about15kDa and39kDa, respectively.2. In vitro, the recombinan proteins of PbCSP-N and PbCSP-N+C could bind tohepatocytes from normal mouse specificallyThe combination of PbCSP-N (or PbCSP-N+C) recombinant protein andmembrane proteins of hepatocytes from normal mice could be recognized by bothanti-His antibody and anti-HSPG antibody. The results showed that PbCSP-N andPbCSP-N+C recombinant proteins could bind to HSPGs on the surface ofhepatocytes.Immunohistochemistry revealed that PbCSP-N and PbCSP-N+C recombinantproteins were markedly localized on the surface of hepatocytes, instead of kidney,stomach, lymph gland, spleen, heart, small intestine, lung and uterus tissues. Theresults indicated that the recombinan proteins of PbCSP-N and PbCSP-N+C couldbind to hepatocytes from normal mouse specifically in vitro.3. In vivo, the recombinan proteins of PbCSP-N and PbCSP-N+C could target toliver from normal miceThe recombinant proteins of PbCSP-N and PbCSP-N+C, Trx protein weresuccessfully labelled with [125I]-sodium iodine. The radioactivities were9.01mCi/ml,13.96mCi/ml,6.76mCi/ml, respectively. Then the proteins and [125I]-sodium iodinewere injected into mice. Compared with Trx protein and [125I]-sodium iodine,PbCSP-N recombinant protein distributed in spleen and liver mainly, andPbCSP-N+C recombinant protein distributed in liver mainly in different time periods,suggesting the two recombinant proteins could target to liver in vivo. Conclusions1. The prokaryotic expressing vectors pET28a-PbCSP-N and pET32a-PbCSP-N+Cwere constructed successfully and the recombinant proteins were expressed andpurified.2. In vitro, the recombinan proteins of PbCSP-N and PbCSP-N+C could specificallybind to the surface of hepatocytes from normal mouse, respectively.3. In vivo, the recombinant proteins of PbCSP-N and PbCSP-N+C could target to liverof normal mouse.