Human Proprotein Convertase Subtilisin/Kexin Type 9 Nanobodies Preparation

The camelid not only has the conventional antibodies that consist of two heterologous heavy and light chains,but also has the heavy chain antibodies that lack of light chains and only consist of two homologous heavy chains.Variable domain of heavy chain of heavy-chain antibodies(VHHs)are the smallest functional antigen binding fragments having been found in nature,also it is named as Nanobody as its size is only 1/10 of conventional antibody.Compared with conventional antibodies,Nanobodies have many unique characteristics such as smaller molecular weight,high solubility and stability,as well as easily to be expressed in bacteria and yeasts.Proprotein convertase subtilisin/kexin type 9(PCSK9)is a secreted serine protease consists of 692 amino acids,which can mediate the degradation of low density lipoprotein receptor and result in increased plasma low density lipoprotein cholesterol level and higher risk of cardiovascular disease.PCSK9 monoclonal antibody inhibitors have been successfully produced and the antibody drugs have only been approved for hereditary hypercholesterolemia market because of its cost.This study aims to identify and produce PCSK9 specific nanobodies through expressing PCSK9 recombinant protein in human embryo kidney 293F cells,immunizing Bactrian camel and identifying the specific PCSK9 nanobodies using high throughput gene and protein sequencing of nanobody repertoire with next generation sequencing and proteomics technology.This approach mainly contains following aspects:1.An expression plasmid has been constructed with pYD7 vector containing the PCSK9 gene and the secretable expression of PCSK9 recombinant protein was successfully achieved in HEK293F cells.The molecular weight of the recombinant protein is about 60 kDa and identical to that of the native human PCSK9 protein.The purity of expressed PCSK9 is about 87.38%and the average expression level reached 4 mg/mL.2.After immunization,the antibody titers and the immune response curves of anti-PCSK9 heavy chain antibodies were measured with an ELISA,and the antibodies titer as high as 1:256000 have been detected after three immunization.Heavy chain antibodies were purified from camel serum from PCSK9 immunized Bactrian camel through a combination using of Protein G and Protein A column,while the magnetic beads conjugated PCSK9 were further used to purify specific anti-PCSK9 heavy chain antibodies which were provided for amino acids sequencing through a proteomics technology.3.After extracting mRNA from PBMCs,multiplex PCR and Illumina Mi-seq sequencing had been used to study the fundamental characteristics of variable domain of heavy chain of heavy-chain antibody repertoire.V and J gene usage,the gene deletion length,V-J pairing preferences,CDR3(complementarity-determining region 3)length distribution,Shannon index,amino acid usage have been intensively analyzed.In average 130000 of useful reads were captured in our Ig-seq runs and 67561 of unique CDR3 gene sequences have been identified.The most frequently used V genes in Bactrian camel heavy chain antibodies are IGHV1S45,IGHV1S50 and IGHV1S52,and that of J genes are IGHJ4 and IGHJ6.More than 40%V-J pairing events were identified in VHH.In summary,a method for rapid preparation of milligram PCSK9 protein in HEK293F cells had been successfully developed,the specific antibody response curves of anti-PCSK9 heavy chain antibodies in the serum of immunized animals had been measured,the specific anti-PCSK9 heavy chain antibodies had been successfully extracted and purified,and finally the fundamental characteristics of Bactrian Camel heavy chain antibodies repertoire had been determined.All these data in this study had established the foundation for identifying and producing the specific anti-PCSK9 nanobodies.