This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S65*01-IGHJ4*01) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×10⁹ clones was obtained, and the titer of the library was demonstrated to be 10¹³ PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.
Single-Domain Antibodies/biosynthesis* ; Peptide Library ; Complementarity Determining Regions/immunology* ; Animals

Objective To compare the efficacy of operative and non-operative methods in the treatment of severe thoracic trauma in Tibetan Plateau.Methods A retrospective case-control study was conducted to analyze 286 patients with severe thoracic trauma admitted to the Shigatse People's Hospital from August 2016 to October 2018.There were 206 males and 80 females,aged 13-71 years [(34.3 ±11.6) years].The duration from injury to hospital ranged from 2 to 49 hours [(22.8 ± 8.3) hours].The causes of injury including fall from height in 109 patients,traffic injury in 98,crush injury in 32,blunt injury in 29,cattle head injury in 9,knife stab injury in 6 and other causes in 3.The injury severity score (ISS) varied from 16 to 48 points on admission [(24.2 ± 8.8) points].A total of 159 patients underwent operation (Operation group) and 127 patients underwent non-operative treatment (Non-operation group).The ISS score was (25.2 ± 8.3)points in Operation group and (23.7 ±7.9)points in Non-operation group.The length of hospital stay,intensive care unit (ICU) care time,complication rate and mortality were recorded in two groups.The correlation of ISS and age with mortality was investigated.Results The length of hospital stay was (12.2 ± 3.8) days in the Operation group and (19.7 ± 5.8) days in the Non-operation group (P ＜ 0.05).In the Operation group,27 patients were admitted to the ICU and stayed for (4.7 ± 1.3)days,and in the Non-operation group 33 patients were admitted to the ICU and stayed for (11.7 ± 3.2) days (P ＜ 0.05).The complication rate was 17.6％ in the Operation group and 31.5％ in the Non-operation group (P ＜0.05).Two patients died in the Operation group,with the mortality rate of 1.3％;six patients died in the Non-operation group,with the mortality rate of 4.7％,with no statistical difference (P ＞ 0.05).There were statistically significant differences in the age of death between the Operation group and the Non-operation group (P ＜ 0.05).The differences in the ISS score and age between the survival and death patients within the two groups were statistically significant (P ＜0.05).Logistic regression analysis showed age (OR =1.090,95％ CI 1.002-1.186) and ISS (OR =1.058,95％ CI 1.027-1.090) were slqnificantly related to mortality.Conclusions For severe thoracic trauma in Tibetan Plateau,operative treatment can shorten the length of hospital stay and ICU care time and reduce the incidence of complications.The mortality ratio of the two groups has no significant difference.Age and ISS may be the high risk factors for death of severe thoracic trauma patients.