Preparation of a New Nanobody and Its Usage in Treatment of Spontaneous Hypertension

Abstract

In order to improve the controllability of spontaneous hypertension, it was necessary to study a new nanobody that can inhibit calcium flow and reduce hypertension by considering the specificity and long half-life of antibody. Firstly, the antibody epitope screening technology was adopted to select a short peptide (CN8) in the E3 region of the first domain of Cav1.2 α1c subunit, the full-length complementary deoxyribonucleic acid (DNA) (cDNA) clone of which was undertaken as a template. The gene fragment in extracellular region of CN8 was amplified by polymerase chain reaction (PCR), and the pET30a-CN8 recombinant plasmid was constructed. Ribose nucleic acids (RNAs) in peripheral lymphocytes of 5 camels were extracted, and the reverse transcribed cDNA was taken as a template to realize the amplification of VHH gene fragment. After the double enzyme digestion, the pMECS vector was connected and TG1 was transformed to form a natural VHH antibody library. The phage display library was screened by CN8 protein to obtain the new nanobody (VHH-CN8). Western blotting (WB) and immunofluorescence (IF) were used to analyze the specific binding of antibody, and the calcium flux test was adopted to test the effect of antibody on calcium channels. A rat model of spontaneous hypertension and a mice model of spontaneous hypertension induced by angiotensin II (Ang II) were constructed to verify the antihypertensive effect of the prepared nanobody materials. In vitro, the VHH-CN8 antibody could realize specific binding to the Cav1.2 channel, inhibit the transfected human embryonic kidney (HEK293) cells, and prevent the increase in intracellular calcium concentration of arterial smooth muscle by adjusting the concentration and potential; in vivo, the VHH-CN8 antibody could reduce the blood pressure level of spontaneously hypertensive rats (SHRs) and mice in Ang II model, which was 196±3/186±4 mmHg (P < 0.05) and 187±5/158±3 mmHg (P < 0.01), respectively. Thus, it proved that the prepared nanobody could reduce the blood pressure level of spontaneous hypertension.