Camelus Bactrianus Natural Nanobody Library Establishment And NGS Sequencing Identification Diversity Application Research

Nanobodies,the smallest functional domains of proteins cloned from the variable region of a special antibody only composed of heavy chains in Camelidae or Chondrichthyes,have broad application prospect.In this research,firstly,the extraction method of RNA samples from Camelidae was explored,and the extraction conditions were optimized;obtained 20 RNA samples of peripheral blood lymphocytes and 1 RNA samples of spleen cells from Camelus Bactrianus with optimized method.Then,four natural nanobody libraries were constructed by cDNA inversion,amplification,conjugation of bacteriophages,electrical transformation and other steps.The capacity and diversity of these four libraries were identified by solid colony counting and high throughput sequencing methods.The results are as follows.1.The results of the RNA extraction optimization experiment show that the A260/A280 and A260/A230 values of the RNA obtained by the two extraction methods are in the normal range;in terms of RNA yield,the precipitation method is superior to the silicon-based membrane method.More than 2 times;RT-PCR detection found that the RNA extracted by the silicon-based membrane method showed the target band.2.The phage display library experiment results show that four antibody libraries CNL1,CNL2,CNL3 and CNL4 were successfully constructed,and the library capacity is good,respectively 4.21 × 109,4.10 × 109,1.74 × 109 and 4.10×109.The insertion rate was verified by random clone sampling,indicating that the insertion rates of the four antibody libraries were all 100%.3.High-throughput sequencing data shows that the ratio of unique sequences to non-unique sequences is 87.70%,89.22%,89.84%,and 86.45%,respectively.Most of the amino acid sequences have hydrophilic mutations at characteristic sites,which is consistent with the stable structure of VHH antibodies Most of the amino acid sequences in the constructed antibody library contain 4 cysteine,and a few sequences are as many as 8-10,which is conducive to the formation of antibody structural diversity.After CD-HIT clusters at a similarity level of 80%,the number of clusters has a large value.The Clustal tool analysis found that the length and distribution of conserved sites are very different.In summary,compared with the precipitation method,the silicon-based membrane method is more suitable for the extraction of camel-derived RNA.Four natural nanobody libraries were successfully constructed using the silicon-based membrane method for library extraction and RNA-binding phage display technology.High-throughput sequencing technology has identified that the constructed natural Nanobody library has good diversity and has great application potential.