The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coli BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77 +/- 5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.
Carrier Proteins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HIV Envelope Protein gp41 ; metabolism ; HIV-1 ; genetics ; Peptides ; genetics ; Repetitive Sequences, Nucleic Acid ; genetics ; Viral Fusion Proteins ; genetics ; Virus Internalization ; drug effects

Aim To examine the effect of zacopride,a specific inward rectifier potassium channel(IK1)agonist,on L-thyroxine(T4)-induced ventricular remodeling and the underlying mechanism.Methods SD rats were randomly divided as control,L-thyroxine(L-thy,1 mg·kg-1·d-1,ig,10 d)model,L-thy +zacopride(5,15,50 μg·kg-1,respectively,ip),L-thy+zacopride(15 μg·kg-1)+chloroquine(7.5 μg·kg-1,ip)and L-thy+captopril(100 mg·kg-1·d-1,drinking water)groups.Echocardiography and cardiac hypertrophic indexes were measured to confirm the establishment of the ventricular remodeling model.The changes of IK1 and L-calcium current(ICa-L)were detected by whole cell patch clamp technique.The confocal microscopy and fluorescent indicator Fluo-4 were applied to examine the intracellular Ca2+ concentration([Ca2+]i)of isolated adult rat ventricular myocytes.Results L-thyroxine induced left ventricular hypertrophy with increased ratio of heart weight(HW)to body weight(HW·BW-1),ratio of left ventrical weight(LVW)to body weight(LVW·BW-1),left ventricular dimension in diastole(LVIDd),left ventricular dimension in systole(LVIDs),interventricular septum thickness(IVS)and decreased ejection fraction(EF),fractional shortening(FS)(P<0.01).Patch clamp data suggested IK1 was downregulated,while ICa-L was upregulated(P<0.01).In isolated adult cardiomyocytes,L-thyroxine increased the cell area and [Ca2+]i(P<0.01).Zacopride treatment obviously alleviated cardiac remodeling,improved cardiac function,reversed the changes of IK1 and ICa-L,and significantly attenuated intracellular calcium overload(P<0.01).The optimum dose of zacopride in vivo was 15 μg·kg-1 at which the effect was compared favourably with captopril,a classical anti-remodeling agent.Low-dose IK1 atagonist chloroquine could reverse the effect of zacopride(P<0.01).Conclusion Via activating IK1,zacopride could significantly decrease Ca2+ influx and intracellular calcium overload thereby inhibiting L-thyroxine-induced cardiac ventricular remodeling.