Prokaryotic Expression, Purification And Polyclonal Antibody Preparation Of Human Placenta Lactogen

Human placental lactogen is a pregnancy-specific hormone which synthesized inplacental trophoblast cells and secreted into the blood of pregnant women,and itsanother name is human chorionic somatomammotropin hormone.hPL-A gene islocated on chromosome17,mature peptide with191amino acids,the molecular weightis about23000Da.It is a polypeptide hormone with two intra-chain disulfide bonds. Inearly pregnancy （5⁶weeks） can be found hPL in maternal serum,when34³8gestational weeks hPL will achieve very high value,since then has been to maintainthe high value to delivery.hPL has important function such as the promotion of breastdevelopment,promoting fetal growth and increase the expression of surface receptorson macrophages.The expression of hPL is relatively decreased,when the placentaoccurred some diseases.Determination of the concentration of hPL in maternal blood,can reflect the the syncytiotrophoblast state,and then judge placental function.Thirdtrimester maternal serum hPL levels can also indirectly reflect the the placentavolume, weight and fetal weight.Measuring hPL expression amount of differentpregnancy period is a trusted reference index,it can monitor high-risk pregnancy,predict the outcome of pregnancy,seize fetal growth and development status and guideclinical diagnosis.Testing pregnant women hPL content has gradually valued byclinicians, which requires a lot of hPL protein standards and anti-hPL antibody.But thesources of natural hPL protein is limited and it’s purification is very difficult.Theprokaryotic expression can be effective to improve the deficiencies.The main contentsand results of this paper are as follows:1Clone hPL gene and prokaryotic expression vector pQE30-hPLPrimers were designed according to hPL gene sequence（NM₀01317.3） providedby GeneBank, using Primer5.0software for the mature peptide gene sequence, in the upstream and downstream primers were introduced by BamHI and HindIII restrictionendonuclease sites.hPL gene encoding the mature peptide region was amplified intotal RNA was extracted from human placenta by RT-PCR technology.2Expression, purification and refolding hPLConstruction of E.coli expression system M15（pREP4）/pQE-30-hPL,then IPTGinduced relative molecular mass of approximately23000Da inclusion body proteinwhich accounted for67%of total bacterial protein.Purification of inclusion body protein denatured by8M urea with SephacrylS-200gel,and the result that the purity is more than90%and the recovery ratereached60%.In order to enable the formation of intra-chain disulfide bonds and restore thespatial structure of inclusion body,the methods of the Cu2+oxidative refolding,ureaconcentration gradient dialysis refolding and column refolding wereapplied.Prokaryotic expression protein after refolding proved to have good biologicalactivity through the colony-forming units-erythroid assay.By Western blot analysis,the purified protein can specific binding withcommercialization of goat anti-hPL,which has good antigenicity and specificity.3Preparation of hPL polyclonal antibodyhPL protein with high purity immunize rabbit to obtain rabbit anti-hPL serumand its titer is greater than1:51200detected by indirect ELISA assay.The rabbitanti-hPL serum can be combined with hPL standard,confirmed that it has a betterreactivity.Prokaryotic expression hPL protein having high immunogenicity becausethe rabbit immunized with a high purity hPL can be obtained with high titer antibody.This study screened M15（pREP4）/pQE-30-hPL strain,it can stably carry therecombinant plasmid pQE-30-hPL and highly express hPL protein.Establishment ofhPL inclusion body protein purification process and showed good biological activityafter refolding.It demonstrated good antigenic and immunogenic function byprokaryotic expressed hPL protein,and by purified and further optimization isexpected as antigen standard.Preparation of the high titer had good specificity ofRabbit anti hPL polyclonal antibody.In conclusion,the paper laid the foundation for the further clinical application of hPL and its antibody.