The Preparation And Application Of Anti-B7-H4 Antibody In Tumor Researches

Background:B7-H4(also called B7S1 or B7x),the newest member of co-stimulating molecules B7 superfamily,is a typeâ… membrane protein encoding 282 aa.B7-H4,discovered by Mayo medical center in 2003,is the third negative regulator of antigen-specific T cell responses and located on choromosome 1p11.1.Genomic DNA of B7-H4 consists of six exons and five introns and spans 66Kb,while the most prominent transcript for B7-H4 is approximately 1.8 Kb.Despite mRNA expression in several human tissues including both lymphoid and non-lymphoid, immunohistochemistry analysis reveal weak positive staining in a little organs from healthy individuals.However,B7-H4 can be inducible expressed on T cells,B cells, monocytes and dendritic cells after stimulation in vitro.In contrast, immunohistochemical analysis demonstrated that B7-H4 was constitutively expressed in freshly isolated cancer tissues such as breast cancer,ovarian cancer and lung cancer. Probing of a mouse tissue Northern blot indicated that mouse B7-H4 has up to four different mRNA transcripts according to the tissue type.The detection of human tissue expressed at least two different transcripts of B7-H4 by RT PCR.These results suggest that B7-H4 expression is tightly controlled in the translational level in peripheral tissues.As an important co-stimulatory molecule,B7-H4 pathway acts by extensive inhibiting CD4⁺ and CD8⁺ T cells proliferation,cell cycle progression,and cytokine production in the process of immune response.B7-H4 may play a role in tumor immune escape machanisms in vivo because it was a negative regulator of cell-mediated immune responses in peripheral tissues and specially expressed in many tumor cells. Therefore,the B7-H4 pathway provides a new opportunity for manipulation of cell-mediated immune responses in cancers,autoimmune diseases,viral infection and transplantation rejection.Blocking the interaction of B7-H4 and its receptor may become a prospective therapeutic strategy of interfering in autoimmune diseases and tumor biotherapy.Objective:In order to study the relationship between B7-H4 and tumor immunity deeply,we designed and synthesized,human B7-H4 polypeptide.Furthermore,we prepared rabbit anti-human B7-H4 polyclonal antibody and studied the difference of B7-H4 expressing in many tumor cell lines and tumor tissues.Methods:1.According to the sequence(AAP37283) publicated by GenBank,we used computer aided design to determine human B7-H4 antigen polypeptide and synthesized it cross-linked KLH(Keyhole Limpet Hemocyanin) so as to increase the immunogenicity of the polypeptide.We used the B7-H4 polypeptide to immunize New Zealand rabbit(initial immunity and four times booster) and obtained rabbit blood serum,purified the anti-B7-H4 antibody in the rabbit blood serum by affinity purification.ELISA(Enzyme linked immunosorbent assay) and Western-blotting were used to identify the valency of anti-B7-H4 antibody and reactive specificity and sensitivity;2.We detected the expression of human B7-H4 protein in multiplicate tumor tissues of tissuechips by immunohistochemistry;3.According to the sequence(NM₀24626)publicated by GenBank,we synthesized the PCR primer of human B7-H4 and detected the expression of B7-H4 mRNA in multiplicate tumor cell lines by RT-PCR;4.We detected the expression of human B7-H4 protein in multiplicate tumor cell lines by Western-blotting.Results:1.Rabbit anti-human B7-H4 polyclonal antibody was successfully prepared;2.B7-H4 protein positively expressed on cytomembrane and cytoplasm of multiplicate tumor tissues(lung cancer,breast cancer,ovarian cancer,uterine endometrioid adenocarcinoma,colon carcinoma,carcinoma of rectum,renal carcinoma) by immunohistochemistry,as well as it expressed on all kinds of normal tissues negatively or weak positively;3.B7-H4 mRNA expressed in eight kinds of tumor cell lines which objective bands were 1200bp around by RT-PCR;4.B7-H4 protein expressed on nine kinds of cell lines by Western-blotting detection. The results displayed that all tumor cell lines(A549,MCF-7,SK-BR-3,CaoV3,Skov3,SiHa,HeLa,HepG2) expressed B7-H4 protein while it was not detected in A293 cell line.Conclusions:1.Rabbit anti-human B7-H4 polyclonal antibody was successfully prepared;2.B7-H4 protein positively expressed on cytomembrane and cytoplasm of multiplicate tumor tissues(lung cancer,breast cancer,ovarian cancer,uterine endometrioid adenocarcinoma,colon carcinoma,carcinoma of rectum,renal carcinoma);3.B7-H4 mRNA and protein expressed on eight tumor cell lines(A549,MCF-7,SK-BR-3,CaoV3,Skov3,SiHa,HeLa,HepG2).