Construction And Expression Of ANTI-HSV-2Single Chain Variable Fragment-Alkaline Phosphatase Fusion Protein

Herpes simplex virus (HSV) is divided into herpes simplex virus type I (HSV-1) and herpes simplex virus type II (HSV-2) two types. They can infect the skin, mucous membranes, as well as a variety of organs. Most genital herpes (GH) is caused by HSV-2infection, but HSV-1has increasingly been shown to also cause genital herpes. HSV-2infection is not curable, mainly through sexual contact horizontal transmission and vertical transmission, highly infectious and can break out repeatedly. Ul-cers produced by herpes episodes is one of the major cause of genital ul-cers. Epidemiological evidence show that HSV-2infection and cervical cancer has a close relationship and can increase the risk of human immu-nodeficiency virus (HIV). HSV-2has a high prevalence and incidence in the crowd, the World Health Organization’s survey data show that the HSV-2infection rate was about16.2%and the HSV-2incidence rate was about0.7%with a rising trend in the population aged15to49years, in2003. Therefore, the establishment of a simple, rapid and low cost of HSV-2screening methods is great significance to the diagnosis and the control of the spread of GH.In this study, the anti-HSV-2scFv gene was cloned into the expres-sion vector pLac-duet-1-AKP containing AKP gene to construct the re-combinant expression vector pLac-H3-AKP to explore the practical value in the diagnosis of GH of the highly specific anti-HSV-2glycoprotein B scFv that previously screened by our laboratory. After confirmed by DNA sequencing, the recombinant vector was transformed into Escherichia coli BL21(DE3) and then induced to express the scFv-AKP fusion protein by IPTG. The fusion protein was analyzed by SDS-PAGE, purified by Ni2+-NTA affinity chromatograph and identified by ELISA after the opti-mal condition for refolding:(50mmol/L Tris-HCl,0.5mol/L NaCl10mmol/L β-mercaptoethanol,0.5mol/L urea,0.5mol/L arginine, lmmol/L GSSG and3mmol/L GSH, pH7.5), the concentration of the re-folding protein is0.13mg/mL,4℃,24h. SDS-PAGE showed that the fu-sion protein existed in a form of inclusion body with an expected mo-lecular mass of about75kD. ELISA confirmed that the fusion protein could bind to HSV-2glycoprotein B antigen and show AKP indicating enzyme activity which provides an effective method for the rapid detec-tion of HSV-2antigen.