Expression And Purification Of Human Anti-HER2 Single-chain Antibody And Study On Proliferation Inhibition Of Tumor Cell T6-17 In Vitro

Objective: To construct prokaryotic expression plasmid of HER2-sc Fv and get the HER2-sc Fv protein in vitro, then to study the inhibitory effect of the HER2-sc Fv protein on tumor cell T6-17 proliferation.Methods : ① In our study, HER2-scFv gene fragment was amplified from plasmid p UC57-HER2-sc Fv by PCR. ②Then HER2-sc Fv gene fragment was cloned into the prokaryotic expression vector p ET28 a through double enzyme, and get the recombinant plasmid p ET28a-HER2-sc Fv. ③ Plasmid p ET28a-HER2-sc Fv was transformed into Rosetta of E. coli. HER2-sc Fv protein expression was induced by Isopropyl β-D-1-thiogalactopyranoside(IPTG). ④HER2-sc Fv protein was purified through Nickel-affinity chromatography column. ⑤ The successful expression of HER2-sc Fv protein was detected by Western blot. ⑥ HER2 positive tumor cell T6-17 proliferation inhibition was tested by MTT assay. MTT assay experiment were divided into 3 groups as follows: HER2-sc Fv protein group, trastuzumab group and control group(PBS).Results : ① HER2-scFv gene fragments were obtained by PCR successfully. ②Location of HER2-sc Fv gene and its sequence in recombinant plasmid p ET28a-HER2-sc Fv was in a right direction by gene sequencing. HER2-sc Fv proteins of about 29 KDa were successfully obtained. HER2-sc Fv protein concentration was 1358.3mg/L, protein purity was about 90% and 10 mg of purified HER2-sc Fv protein per litre can be received. ③ MTT assay showed that HER2-positive tumor cell T6-17 proliferation inhibition increased in HER2-sc Fv groups(33.52%), compared with that in PBS groups, difference was statistically significant(P=0.000, P <0.01). Trastuzumab group(34.79%) also had a higher inhibition of proliferation in trastuzumab group than that in PBS group(P=0.000,P<0.01). But there was not significant statistical difference between HER2-sc Fv group and trastuzumab group(P = 0.429,P> 0.05).Conclusion: ① Recombinant plasmids pET28a-HER2-sc Fv are constructed successfully. ② HER2-sc Fv proteins have been successfully expressed and purified by IPTG induction and affinity chromatography. ③ HER2-sc Fv proteinssignificantly inhibit of HER2-positive tumor cell T6-17 proliferation in vitro.