Expression Of Human Platelet Factor 4 In Cultured Cells And Larvae Of Bombyx Mori

Human platelet factor 4 (hPF4) is a protein found in megakaryocytes and plateletα-granules. It is a typical protein of multiple biological functions. The functions sofar identified include antitumor, neutralizing anicoagulant activity of heparin,immunological regulation, inducing inflammation reaction and repairing wound etc.Extensive studies on hPF4 have been made in recent years and more and moreattention has been paid to its basic and clinical iatrology. In this study, therecombinant hPF4 was expressed in Escherichia coli and in baculovirus expressionvector system.The hPF4 gene was inserted into the multiple cloning site of the expressionvector pBV221 to construct a recombinant expression vector pBV-PF4 andoverexpressed in E. coli JM109 under the control of the bacteriophage λPRPLpromoter regulated by a temperature sensitive repressor. The expression products wasidentified by Western blotting and ELISA, in both reactions the thPF4 activity wasdetected. At the same time, a protein band of about 8kD, which is similar to themolecular weight of natural PF4(7.8kD), appeared on the SDS-PAGE profiles. About9.968 μg/ml was detected in E. coli culture by ELISA.Then, the hPF4 gene was overexpressed in Bombyx mori baculovirus expressionvector system for the first time. The recombinant transfer vector pBacPAK-PF4 wasconsmtructed by insertion of hPF4 gene into the transfer vector pBacPAK 8 and used tocontransfect the BmN cell line with linearized baculovirus BacPAK6 DNA under themediation of Dosper agent. Then the recombinant baculovirus BacPAK-PF4 wasidentified by plaque assay DNA dot blotting and southern hybridization showed thatthe baculovirus contains the hPF4 gene. BmN mono-layer culture cells (2×106/flask)were infected with the recombinant virus BacPAK-PF4 at a MOI of 10. Transcriptionof PF4 mRNA was detected by Northern blotting. Activity assays of expressionproductS on enthelial cells ECV304 showed activities of hPF4 and the amoun ofexpressed hPF4 in extract of cultured cells reached the maximum level at the therd daypost-infection (6.088 ll g/2X 10'cells) and the inhibition percent tO the growth ofenthelial cells ECV304 reached 90.6%. Westem blot and ELISA detection of theexpression product also showed the activity of hPF4 and two protein bands of about8kD and l7kD aPpeared on the Westem blot profiles. ms imPlied that the expressedhPF4 may be in tWO forms either as mature protein or in itS unprocessed fOrm. The relationshipbetween exPression amount and MOI was also determined. The resultS showed that in cell eXtractthe activity was increased in a logarithmic manner when MOI was increased from 0. l -l0.hPF4 was also expressed in si1kWorm larvae and puPa. The activity assaysshowed similar results in both of them. Maximurn activity aPPeared at the fiffe daypoSt-infection and from then on the activity was declined, implying that the best timefor collecting the hemolymph is at the fifth day The expression axnoun in hemolymphreached 29.02 ll g/ml and the inhibition percent of ECV304 growth reached 92.3% atthe fifth day Westem blot and ELISA results were similar to that in BmN cells.Westem blot also showed twO protein bands at the position about 8kD and l7kD. Itrevealed that hPF4 may also be expressed in two forms in hemolymph either as matureprotein or in its unprocessed fOrm.Furthermore, the expression efficiency among silkworm larvae, BmN cells and Ecoli was compared. The expression amoun in hemolymph is much higher than that inBmN cells and E coli. The activity assay showed a maximum expression of 29.02 llg/ml in larval hemolymph which is 2.9-fold higher than that expressed in E coli. Theexpression amount in one larva was equivalent to that in 6 flasks of BmN cells inrespect of total biological activities.