hiv自然感染与疫苗诱导产生抗体的快速鉴别诊断试剂盒研制

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1、重庆医科大学硕士研究生学位论文PARTIANALYSISOFBIoLoGICALACTIVITYOFHIVPEPTIDESoBTAINEDBYPROKARYoTICEXPRESSIONANDCHEMICALSYNTHESISObjective：ToobtainHIV-1antigenpeptidesthatcoulddifferentiatevaccine．．inducedantibodiesandthosefromvirus—inducedMethods：1．prokaryoticexpression：Theg

2、enesofskl，sk2，sk3andskl—sk2一sk3wereamplifiedbyPCRandoverlapextensionPCR，thenrespectivelyinsertedthetargetgenesintotheexpressionvectorspET32a(+)andpGEX4T-2．TherecombinantvectorsweretransformedintoE．coliBL21(DE3)andinducedbyIPTG．ThefusionpeptidescontainedHisorG

3、STtagwerepurifiedbyNi-affinitychromatographyorGST-affinitychromatographyandanalysedbySDS—PAGE．TheantigenicityoftheseproductswasassayedbyimmunoblotusingHIVpositiveserum．TheELISAkitswerepreparedwiththeserecombinantpeptidesandtheperformancesofthekitswereevaluate

4、dbyclinicalsamples．2．chemicalsynthesis：HIV-1peptideswereobtainedbywayofchemicalsynthesisandanalysedforantigenicitybydot-blot，andthespecificityandsensitivityofELISAkitswereanalysedbyclinicalsamples．Result：1．prokaryoticexpression：His-skl、His-sk123、His—sk213、His

5、—sk231andGST-sk3、GST-skl23、GST-sk213wererespectivelyexpressedinpET32a(+)andpGEX4T02．HIV-tpositiveserumandHIV9重庆医科大学硕士研究生学位论文DNA-Tiantanvaccineserumwesternblotingprovedthatalloftheseexpressionproductshavethebiologicalactivity．WhenthesefusionpeptidesusedinELISA

6、kits，thesensitivitywaslessthan50％andthespecificitywaslessthan90％．2．chemicalsynthesis：Theresultofdot-blotshowedthatchemicalsynthesispeptidesklwellreactedwithHIV-1positiveserum，sk2andsk3havelowerreactivity．Synthesispeptidesdidn’treactwithHIVnegativeserumsamplea

7、ndHIVTiantanvaccinesinducedserumsample．TheELISAresultsshowedthatthreepeptideshad92％sensitivityand100％specificityindetectionofclinicalsamples．Conclusion：ThreeHIVpeptideswereobtainedwhichhaveexcellentbiologicalactivityandcouldbehopefullyappliedinHIVdifferential

8、diagnosiskit．Keywords：HIV,polypeptide，recombinant，chemicalsynthesis重庆医科大学硕士研究生学位论文PARTⅡDEVELoPMENTOFHIVRAPIDDIFFERENTIALDIAGNOSISⅪTObjective：Todevelopanovelrapidflow—throughtestkitusingth