肿瘤特异性蛋白B7-H4纳米抗体的筛选和鉴定

Screening and Identification of Nanobody for Tumor Specific Protein B7-H4

羊驼体内存在天然缺少轻链的重链抗体,克隆重链抗体可变区(VHH),即可构建单域抗体(single-domain antibodies,sdAbs),又称纳米抗体(nanobody,Nb)。利用非免疫羊驼噬菌体文库筛选肿瘤特异性蛋白B7-H4的纳米抗体,经过4轮淘选,ELASE鉴定阳性克隆噬菌体,测序获得其DNA序列后体外转录为mRNA,将修饰纯化后的mRNA转染到肿瘤细胞,利用细胞免疫荧光检测mRNA在肿瘤细胞内是否表达,Western印迹进一步验证其表达情况;通过CCK-8法鉴定其对肿瘤细胞的增殖抑制能力;划痕实验鉴定其对肿瘤细胞迁移能力的影响;Transwell法鉴定其对肿瘤细胞的侵袭能力的影响;裸鼠荷瘤模型瘤旁注射mRNA,鉴定其在体内实验对肿瘤组织的作用。结果显示,通过淘选获得1个高亲和性的噬菌体株菌H6;DNA测序并导出的氨基酸序列符合羊驼纳米抗体结构;将其mRNA导入肿瘤细胞,能有效表达出纳米抗体H6;转染H6-mRNA的肿瘤细胞(0.84±0.08)与未转染H6-mRNA的对照组(1.83±0.04)相比,其增殖能力明显受到抑制,P<0.01,其迁移和侵袭能力(78.60±5.36)明显低于空白对照组(197.80±21.04),效果优于B7-H4 mRNA的siRNA(95.40±16.56);在裸鼠乳腺癌模型中能有效抑制肿瘤生长,效果优于紫杉醇和B7-H4 mRNA的siRNA。这说明筛选所得抗B7-H4纳米抗体H6能特异结合B7-H4蛋白并封闭其功能,导致肿瘤细胞的增殖、迁移和侵袭受到抑制。该结果为利用B7-H4作为治疗癌症的靶点提供了实验基础。

Single-domain antibodies(sdAbs),in particular those engineered from the variable heavychain fragment(VHH gene)found in Camelidae heavy-chain antibodies(or IgG2 and IgG3),are the minimal fragments that retain the full antigen-binding capacity of the antibody.A phage-displayed random non-immune alpaca library was used to screen B7-H4 nanobody for four rounds of affinity screening.ELISA was used to test the binding affinity of the selected phages to B7-H4.Its DNA sequence was obtained and sequenced In vitro transcription to mRNA.Modified mRNAs are particularly useful for transient expression of proteins but also can be used for stable expression via repeated transfections.The proliferation of cancer cells was determined by Cell Count Kit(CCK-8)assay and wound healing assay.The invasion of cancer cells was detect by Transwell assay and the levels of B7-H4 nanobody verified by cell immunofluorescence and Western blotting.The effect of in vivo injection of mRNA on tumor tissue after tumor bearing was identified in nude mice.Sequencing results showed the highest repeat sequences was H6.The small molecular oligonucleotide H6 that can specifically bind to human cancer cells is successfully screened in vitro using phage-display technology.After retrieval,the peptide segment is not homologous with known proteins.H6-mRNA was successfully transfected into cancer cells.Cell immunofluorescence and Western blotting results showed that H6-mRNA transfection inhibited the expression of H6-protein in cancer cells.The proliferation of cells transfected with H6-mRNA(0.84±0.08)were significantly lower than in blank group(1.83±0.04,P<0.01).The number of invasive cells in the H6-mRNA group(78.60±5.36)was significantly lower than that of the blank control group(197.80±21.04),and siRNA group(95.4±16.56)targeting B7-H4-mRNA.It can effectively inhibit tumor growth in nude mouse breast cancer model and the effect is better than paclitaxel and siRNA targeting B7-H4 mRNA.The screened anti-B7-H4 nanobody H6 can specifically bind to B7-H4 protein and seal its function,leading to the inhibition of proliferation,migration and invasion of tumor cells.So it should be likely to become a new target for treatment of cancer.