Construction of in vitro high-throughput screening system for activin receptor-like kinases 4, 5 and 7 inhibitors / 中国药理学与毒理学杂志

OBJECTIVE To establish an in vitro screening system for activin receptor-like kinase 4,5 and 7 (ALK4,ALK5 and ALK7) inhibitors.METHODS The insect expression systems for kinase domain of ALK4,5,7 and Smad2/3 proteins were established using the Bac to Bac baculovirus expression system.The desired proteins were expressed in Sf9 insect cells and purified by GST affinity.The screening system was composed of the kinase,Smad3 protein,ATP as well as the compound.The impact of the compound on the activities of ALK kinase domains was examined by measuring the amount of remnant ATP in the system as ALKs catalyzed the phosphorylation of Smad3 protein and consumed ATP during the process.The screening conditions were optimized,and validation of the screening system was conducted using known ALKs inhibitors.RESULTS All the reconstructed Bacmids were identified to be correct by PCR and restriction enzyme digestion.All the proteins were expressed in Sf9 insect cells after transfection,and purified proteins were achieved by GST affinity purification.For the screening system,the optimized kinase concentration and Smad3 concentration were 10 mg· L-1 and the optimized ATP concentration was 10 nmol·L-1.The Z'factor for ALK4,ALK5,and ALK7 kinase inhibitors screening system was 0.71,0.51 and 0.74,respectively.The well-known ALK inhibitor SB431542 inhibited the catalytic activities of ALK4,ALK5,and ALK7 with IC50 values of 22,188 and 91 nmol· L-1,respectively.CONCLUSION The in vitro screening system for ALK4,ALK5 and ALK7 inhibitors is successfully established.

Expression and purification of autotoxin in sf9 insect cells / 中国生化药物杂志

Objective Production of autotoxin protein in sf9 insect cells with biological activity. Methods Autotaxin cDNA was cloned into pFastBacTMHTA from melanoma cell by extraction of total RNA using TRIzol method and RT-PCR. Bacmid-ATX is isolated from transformed competent bacterial DH10 which carries Bac genomic sequences and transfected into sf9 using lipofectamine 2000. Recombinant ATX virus was amplified in sf9 and further used for infection and expression of ATX protein. Two step purification product using HistrapTMHP and Hiload 16/600 Suerdex 200pg was determined for lysophospholipase D (lysoPLD) activity. Results Correct insertion of PCR fragment is confirmed by BamH I/Xho I digestion and sequencing. ATX virus can infect sf9 and induced enzymatic activity. Column purification and SDS-PAGE resulted 95% in purity and 6mg/liter in yield with significant lysoPLD activity. Conclusion ATX Baculovirus was successfully constructed that can infect sf9 cells and express active lysoPLD. Production of active ATX can be used for crystalography studies and screening for small pharmaceutical inhibitors.

Construction and identification of KIF18A protein baculovirus expression system / 国际生物医学工程杂志

Objective KIF18A is a protein that has close relation with mitotic regulation and tumor development.This study aimed to establish KIF18A protein expression system in baculovirus,which may help to realize high efficient synthesis of KIF1 8A protein in vitro.Methods Transfer vector was constructed with molecular cloning method,and recombinant baculovirus were obtained through gene transposition.KIF18A protein was expressed by transforming recombinant baculovirus into infected insect Sf-9 cells to realize the synthesis efficiency.Results It was confirmed by DNA sequencing,microscopic observation and Western Blot that the KIF18A protein expression system in baculovirus was successfully established.Conclusions Conditions for transfer vector and recombinant virus transfection are defined,and high efficient KIF18A protein baculovirus expression system are successfully constructed.

Expression of soluble H1N1 haemegglutinin in two distinct insect baculovirus expression systems / 中华微生物学和免疫学杂志

Objective To express functional haemegglutinin(HA)protein in two different bacularvirus expression systems.Methods The whole open reading frame of A/Sichuan/1/2009(H1N1)HA was obtained by synthesis,and the HA protein were expressed in insect cells by two different bacularvius expression systems:BaculoGold system and Bac-to-Bac system. Soluble HA protein was identified by Western blot and haemegglutination test. Results The correct full length of HA gene was obtained and cloned into pAcGP67B and pFAST Bacl vectors,respectively.After 3 rounds of virus amplifyjng by re-infection of Sf9 cells,the HA protein was detected in supematant of BaculoGold system and in intracellular of Bac-to-Bac system which is much better than the former.Purified HA protein was positive not only identified by Western blot,but also detected by haemegglutinin test. Conclusion Functional HA protein was successfully expressed in two distinct bacularvirus expression systems,of which the Bac-to-Bac bacularvirus expression system is more suitable for expression of A/Sichuan/1/2009(H1N1)HA protein.

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru / 中国病毒学

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.