| HCV is a non-A, non-B hepatitis virus, and was named the hepatitis C virus (HCV), belonging to Hepacivirus, Flaviviridae.Hepatitis C virus is a major liver disease that jeopardizes the world’s population. Hepatitis C is caused by the infection of hepatitis C virus (HCV) mainly by the ways of transfusions, acupuncture and drug abuse. According to World Health Organization statistics, more than170million people are infected with hepatitis C virus worldwide. Just in China there are more than fourteen million Chinese have HCV infection, the infection rate is as high as3.2%, which means our country is a high-risk area of HCV infection. Approximately50-80%of HCV infected individuals spontaneously become chronically infected, and10-20%of which are further at risk of developing chronic liver disease including liver failure, liver cirrhosis,1-5%of the patients developed into liver cancer in three decades later.Hepatitis C virus is a member of Flaviviridae virus family. HCV virus is generally spherical, and HCV genome is a single-stranded positive strand RNA molecule, approximately9.6kb, which is composed of multiple copies of small basic protein, including the structural protein, non-structural protein and non-coding areas. It has one large open reading frame that encodes for a poly protein of3,011amino acids, which can be cut into10HCV proteins, still having their individual functions, containing structural protein core (C), envelope1and2(E1,E2), p7, and nonstructural protein NS2, NS3, NS4A, NS4B, NS5A, NS5B, by host and viral proteases.HCV core region is not only important to gene and serum HCV genotyping for targeting gene region, but also to the treatment of HCV infection on gene level as well as to the vaccine research. Because the HCV core region is the most conservative gene region of the HCV and it encodes predominant antigen which has the strongest immune reaction.Nonstructural protein3(NS3)(1192-1457aa) provides protease, helicase and NTPase enzymatic activities that play a crucial role in viral replication.The N-terminal third of NS3contains a serine-protease domain responsible for processing the nonstructural polyprotein of HCV, while the C-terminal two-thirds encode for an adenosine triphosphatase (ATPase)/helicase capable of unwinding duplex RNA. Previous studies revealed that the NS3helicase contains immunodominant B-cell epitopes eliciting high levels of antibodies in HCV infected individuals. The human and murine humoral immune responses to HCV NS3protein are almost exclusively targeting the ATPase/helicase domain, which appears to be serologically reactive during the early phase of HCV infections and is routinely used in clinical diagnostic HCV antibody immunoassays.Currently there is still lack of effective rapid diagnostic reagents to detect hepatitis C virus infection. In China, screening of blood donors infected with the method of HCV Elisa detection, which using two-pass rate to screen the blood donors infected, in line with fake positive rate as high as30percent, causing great damage to the blood donors.The current inspection methods use recombinant protein Core, NS3, NS4, and NS5expressed in E.Coli as antigens to detect HCV antibody. This method is of weak reactivity and specificity, seriously affecting the accuracy of the HCV antibody detection. However, using the Eukaryotic expression system to express HCV antigen will improve the efficiency of detection. The protein-modification process can be more conducive to its processing chains of amino acids folded into the native conformation of the protein, and thus brings higher specificity. And proteins expressed in eukaryotic expression systems have lower unrelative immunogenicity, the risk of non-specificity from prokaryotic expression system can be much reduced. Huh7.5.1cell lines, derived from human hepatoma cell line, was used for in vitro studies of HCV as one kind of major cell lines. Compared to other tissue cell lines, it is easier to express HCV-related proteins and the protein processing methods are closer to the folded conformation of the native proteins, which means a lot to the expression of native and high-activity HCV Core and NS3protein.Lentivirus (LV) is a member of retro viruses, converted from the HIV-1lentiviral vector system. It has gradually become researchers’primary choice for its high efficiency and stability in gene transfer in recent years. Besides, the Lentivirus can infect both mitotic cells and non-dividing cells, and the gene carried by the LV can be integrated into the host genome and can offer stable long-term transgene expression while resisting to the transcriptional silencing effect of the host genome. Meanwhile, the process that LV infects target cells with the HCV virus is similar to the HCV natural infection, but the virus can not replicate automatically, which brings much higher biological safety.In summary, compared with other retroviral vectors, LV has a high transduction efficiency, and the proteins expressed are more stable and of higher biological safety. Therefore, lentiviral vectors can be a powerful tool for transgene delivery into cells to express recombinant HCV Core and NS3gene. Thus it is of great significance and advantages that packaged recombinant lentivirus with target gene infect cells can promote the expression of protein Core and NS3antigens. We obtain the gene copies of Core and NS3by copying the plasmid SQT7DH-5, which contains the whole lb subtype HCV Core gene, and the plasmid pMD20NS2-4A, containing NS3gene. Meanwhile, we introduce hydrophilic signal peptide SP (MWTLVSWVALTAGLVAG) into the N-terminal, and his-tag into the C-terminal, of protein Core and NS3by the means of primer design. Which can be shown as, from the N-terminal to C-terminal, SP-Core/NS3-6His. Besides, we insert our target sequence into the Lentivirus vector pHAGE-fullEF1alpha MCS-IZsGreen, to build the expression vector pHAGE-fullEF1α-Core-MCS-IZsGreen and pHAGE-fullEF1α-NS3-MCS-IZsGreen. Using the method of liposome transfection, we infect the293T cells with a certain scale of packed Lentivirus vectors and packing-auxiliary plasmid pMD2G and psPAX2. Through the repackage of293T cells, we get infectious recombinant lentivirus LV which has integrated with HCV Core and NS3gene, the LV-Core and LV-NS3. We infect the huh7.5.1cells, a kind of human liver cell line, with a certain amount of LV-Core and LV-NS3, and find that its RNA can reverse the transcription into cDNA and integrate with garget cell genome specifically, promoting the expression HCV Core and NS3antigen in Huh7.5.1cells. Because proteins expressed in Huh7.5.1are hydrophilic protein, for its six-histidine tag, which can be purified through the Ni-NTA affinity chromatography, we can get the purified NS3by easily. Core is a strongly basic protein, to obtain purified Core protein, we can screen the protein by the method of cation exchange. Incubate the diluent Core and NS3on specialized slat as HCV ELISA detective materials to detect the28serums, which have been tested to be HCV antibody positive samples from the shenzhen blood center, so that we can evaluate the sensitivity and specificity of this novel HCV antigens.Through the experiment mentioned above, we build the expression vector, containing the purpose protein gene pHAGE fullEF1α-Core-MCS-IZsGreen and pHAGE-fullEFl α-NS3-MCS-IZsGreense by the means of conventional molecular cloning, and preliminarily identified that these two plasmids can correctly express target proteins and labeled proteins in eukaryotic cells,293T, by using the method of transient transfection, but the target protein can not be secreted to the outside of the cells. By common transfection of packed-expression plasmid and the third generation of Lentivirus in two other packing-auxiliary plasmid to293T, the gene copy number becomes2.3×109copies/ml LV-and the Core virus gene copy number is3.3×108copies/ml LV-NS3. By infecting Huh7.5.1cells and293T with a certain amount of recombinant Lentivirus respectively, and observing the green fluorescence-labeled protein in Huh7.5.1and293A cells, we find that Huh7.5.1is more likely to get infected by recombinant Lentivirus than293A cells. According to immunofluorescence test, recombinant NS3and HCV Core protein are mainly distributed in the cytoplasm, so after cracking the cells, we collect the cracking supernatants and cellular debris on precipitation respectively to perform Western Blot, and found that the Core and NS3protein are partly soluble, and NS3has a better hydrophilic than Core has.This paper use a eukaryotic expression system, the novel lentiviral-mediated expression system, which works similar to HCV replication mechanism, to produce Core antigens and set up a method fo largely produce and purify Core protein. This expression system takes both the advantage of LV vector and the eukaryotic expression system, and may provide us novel antigens for infection test and clinical diagnostic immunoassays. |
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