ObjectiveTo humanize the variable region of the IL9-2G1 rabbit monoclonal antibody （IL9 2G1 mAb） and to evaluate its biological activity. MethodsThe humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region （VH） and light chain variable region （VL） of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell （CHO） expression system. Enzyme-linked immunosorbent assay （ELISA） was used to qualitatively analyze the antigen-binding activity of the antibodies. After screening the optimal humanized antibody candidate， surface plasmon resonance （SPR） was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 （JAK1/STAT3）， while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 （IL-9） secretion. ResultsPurification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant （KD） of M501042-OHFL against the analyte M30903 was 1.33×10^-7 M， which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 （p-JAK1） and phosphorylated STAT3 （p-STAT3） were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group （P<0.01）. The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group （P<0.01）. ConclusionHumanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody， and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed， which provides an experimental basis for subsequent research.

Objective : To construct triggering receptor expressed on myeloid cells 2(TREM2) gene knockout(TREM2-/-) mice using CRISPR/Cas9 technology, to breed TREM2-/- mice and to analyze the genotype of TREM2-/- mice. Methods : CRISPR/Cas9 technology was used to selectively knock out exon 2-3 regions of TREM2 gene to construct a TREM2-/- mouse model, and the genetic background of all mice was C57BL/6J. Polymerase chain reaction(PCR) was used to identify the genotype of mice. Quantitative real-time PCR(qPCR) and Western blot were used to detect the expression level of TREM2 in major tissues of mice, and the authenticity and scientific nature of PCR identification results were verified from mRNA level and protein level. According to the sgRNA sequence, the possible off-target sites were predicted on the CCTop website, and the tail DNA of mice was extracted and amplified by PCR and then Sanger sequencing was performed to detect whether there was off-target effect in TREM2-/- mice. Results : TREM2-/- mice were successfully constructed by CRISPR/Cas9 technology, and the mice were genotyped. The results of agarose gel electrophoresis showed that the mouse genotype with only 415 bp band amplified was wild type(WT), the mouse genotype of the 449 bp band amplified only was TREM2-/-, and the mouse genotypes amplified with 415 bp and 449 bp double bands were heterozygous. qPCR results showed that compared with WT mice, the mRNA expression of TREM2 in heart and brain tissues of TREM2-/- mice was down-regulated(P-/- mice was reduced(P-/- mice. Conclusion TREM2-/- mice are successfully constructed and bred, a reliable genotype identification method is established, the genetic stability of the mouse model is verified, which will provide an important genetic animal model for the study of TREM2 gene function.

Objective:to investigate clinical significance of serum and urine NGAL (Neutrophil gelatinase-associated lipocalin) in the early kidney damage with primary hypertension.Methods:According to UAER ( urinary microalbumin excreting rate ) ,we divided 90 patients with primary hypertension into three groups (<30 mg/24 h,30-300 mg/24 h and >300 mg/24h),and selected 30 healthy people as the control group.Serum and urine NGAL and cystatin C ,serum creatinine,urea nitrogen,high sensitive C reactive protein , transferrin,and basis of blood pressure were detected and followed up one year.Results:Compared with healthy group ,GFR( glomerular filtration rate ) and serum NGAL were decreased significantly in medium and severe proteinuria groups , while urine NGAL was increased.Conclusion:Serum and urine NGAL have been a clear trend changes in kidney damage ,which could be used as a reliable indicator in monitoring renal function of patients with primary hypertension.

Objective Severe acute respiratory syndrome is recently emerged infectious disease caused by a novel coronavirus, but its immmunopathological mechanism has not yet been fully elucidated. Method We established the SARS animel model and investigated changes in plasma inflammatoy cytokines monkeys and rats. 8 monkeys with PCR and antibody positive were detected. Serum levels of inflammatory cytokines interleukin(IL)-6,IL-10,Th-1 cytokine,interferon(INF)-? and tumor necrosis factor(TNF)-? were measured by enzyme-linked immunosorbent assays(ELISA).Result The concentration of IL-10 and TNF-?were not significantly different in model and control group.IL-6 showed marked elevation of at least 10 days, and there was a positive relationship between the level of IL-6 and pulmonary pathological changes. The INF-? level decreased. Conclusion The result of sera level of SARS animal model avoid the disturbance of anti-viral drug and corticosteroid, it suggest that there are different immunoregulatory events during SARS and may contribute to our understanding of the pathogenesis.