Construction And Expression Of The Recombinant Adenovirus With L1 Gene Of Human Papinom-avirus Type16,Zona Pellucida 3 Gene Of Mouse And Lagurus Lagurus

1. Construction and Expression of the Recombinant Adenovirus with L1 Gene of Human Papillomavirus Type 16 Human papillomavirus (HPV)is a double strand DNA virus. HPV with high risk, such as the familiar the most type16 and type 18, can lead to the malignant tumor, e.g., cervical carcinoma. There are large amounts of data identified that 50 percent of cervical carcinoma is due to the infection of HPV16. It is not an appropriate method to develop an attenuated vaccine or inactivated vaccine of HPV because it contains DNA which has potential carcinogenesis. It is now focused on the subunit vaccine of later protein L1 and L2 to develop HPV16 vaccine.The later gene L1 of HPV16 codes for major capsid protein in virus. HPV16 L1 or HPV16 L1+L2 produced in eukaryotes can automatically and efficiently assemble to virus like particles. The VLPs become a concerning immunogenic material because it doesn't contain DNA and shares the similarity with the crude virus in structure , therefore , it can stimulate organism to secrete antibody to prevent human from infection by same type of HPV , so L1 protein becomes an important target antigen to produce HPV vaccine. HPV16 L1 gene which was previously acquired from cervical carcinoma tissue of Xinjiang Uygur women in our lab occurs variation multiple sites hence forming the main mode of mutations. The mutation can cause the hydrophobic and antigenic changes of HPV16L1. The mutation in L1 gene will give rise to HPV16 escaping from immunity recognition which can cause continuous infection of HPV16. Research in DNA vaccine aimed at HPV16 L1 in xingjiang will greatly protect people from HPV16 infection.The HPV16 L1 gene cloned previously by our lab from cervical carcinoma tissue of Xinjiang Uygur women, was subcloned into shuttle vector pShuttle, a two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAd-L1, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. PCR test indicated that L1 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.5×10~9 pfu/L, meanwhile, RT-PCR demonstrated the transcription of L1 in infected cells, Western blot and IFA revealed that L1 protein can express in HEK293 cells, which will be potentially used as a candidate vaccine for the prophylaxis and treatment of HPV infection.2. Construction and Expression of the Recombinant Adenovirus with Zona Pellucida 3 Gene of Murine and Lagurus lagurus The mammalian oocyte is surrounded by an extracellular translucent envelope termed the zona pellucida (ZP). The ZP mediates the initial recognition and binding events of sperm to the oocyte in a relatively species specific manner, thus playing a vital role in fertilization. In most species, ZP is composed of three biochemically and immunologically distinct glycoproteins, ZP1, ZP2, and ZP3, which can recognize and combine with sperm and induce acrosome reaction. It has been widely concentrated that fertilization could be successfully prevented when the combination of ZP3 and sperm was blocked by monoclonal antibodies both in vivo and in vitro, and so ZP3 glycoprotein has been considered a good candidate for developing an immunocontraceptive vaccine.Recombinant adenoviral vectors can imitate virus inartificial infection manners, delivery the foreign genes effectively to host cells, then express natural antigens with full bioactivity , which can induce not only effective and specific humoral but also intense cell mediated immune responses. The most commonly used adenoviral vector is human adenovirus serotype 5, which is rendered replication defective by the deletion of the E1 and E3 genes. Not only do these deletions render the virus incapable of replicating itself, but they also create space for up to 7.5 kb of foreign DNA. Recombinant adenovirus can infect both dividing and non-dividing cells, and produce high titers as well, the recombinant adenovirus remains epichromosomal in the human host cell, there is no possibility of activation or inactivation of host cell genes resulting from interruption by the transfected gene(s),so replication defective adenovirus vectors have distinct advantages as an immunocontraceptive vaccine vectors.The lzp3 and mzp3 genes cloned by our lab from Lagurus lagurus and mouse , were subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with lzp3 and mzp3, which were digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. PCR test indicated that zp3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2×109 pfu/L and1.3×109 pfu/L, IFA revealed that ZP3 protein can express in HEK293 cells. Then HeLa cells were infected by the recombinant adenovirus. RT-PCR demonstrated the transcription of zp3 in infected cells; Western blot revealed that the recombinant adenovirus RAd-lzp3 and RAd-mzp3 can be successfully expressed in the infected HeLa cells, which lays the foundation for developing a new immunocontraceptive vaccine against Lagurus lagurus and murine zona pellucida 3.