Construction Of Fully Human Anti-her2 Monoclonal Antibody Transgenic Goat

According to CA Cancer J Clin’s article,the breast cancer ranks first in the incidence of female malignancies,and second only to lung cancer in the global cancer incidence,among the global incidence of cancer,in 2018.The breast cancer has become the first major malignant tumor that harms women’s health.Her2-positive metastatic breast cancer(MBC)accounts for 25%of advanced breast calcer.Human epidermal growth factor receptor 2(Herb/ErbB2)is a transmembrane protein with tyrosinase activity.The overexpression of Her2 is usually related to the deterioration of many epithelial cancers.Using Her2 as a target site to treat breast cancer has become a research hotspot in the field of cancer therapy.So far,monoclonal antibodies,bispecific antibodies,and antibody-conjugated drugs are mainly included in Her2 antibody-targeting drugs.The US FDA approved the first monoclonal antibody drug trastuzumab(Herceptin?)for the clinical treatment of Her2-positive metastatic breast cancer(MBC)in 1998.To ensure correct modification and biological activity of the antibody proteins,mammalian cells with natural active proteins are generally selected as expression vectors to produce traditional monoclonal antibodies.In this study,the transgenic goat mammary gland bioreactor was chosen as antibody expression.The pDBC-Her2 was constructed as the anti-Her2 mammary gland-specific expression vector,whose expression contains two goat B-casein as promoter and two bovine BGH-PA as terminator frame.The light chain and heavy chain of the Her2 antibody were ligated into two expression cassettes respectively,and the expression vector was able to integrate the foreign aid gene into the human lysozyme transgenic goat cells through the Cre/loxp recombinase system.After transfecting primary goat mammary epithelial cells,detecting the cell-level expression of cDNA and protein by RT-PCR and Western Blot,and the construction of anti-Her2 mammary gland specific expression vector pDBC-Her2 was completed.The constructed pDBC-Her2 plasmid and pBS185 plasmid were co-transfected into human lysozyme goat fibroblasts,and positive clones were obtained by pressure screening of puromycin.PCR and sequening were performed to verify whether the target gene was integrated into a predetermined site and the target was successfully obtained.The integrated positive cell strain is also a somatic cell nuclear transfer donor for the preparation of transgenic goats.30 healthy female Guanzhong goats aged 2 years old were selected,of which 20 were donors and 10 were recipients,controlling simultaneous estrus.248 oocytes in total were obtained after superovulation and surgical retrieval.92 embryos were reconstructed and transplanted into 10 recipient ewes by surgery.B-ultrasound was performed 35 days later after transplantation,and 3 ewes appeared in the gestational sac.The pregnancy rate is 20%(2/10).The 40-day-old sheep fetus was removed by laparotomy and identified by PCR and sequencing.The fetal gene was successfully localized and integrated with the Her2 antibody gene.The pDBC-Her2 transgenic sheep was constructed,which laid the foundation for the subsequent expansion of the transgenic sheep.