Table 1

Primers for qPCR"

Fig. 1

Expression of Qa-1b in skins of Mongolia cattle A. Results of the mRNA expression of Qa-1b in the skin, Y is calf, M is adult samples; B. Results of the protein expression of Qa-1b in the skin, 1-3 is calf, 4-6 is adult cattle; C. Grayscale value analysis; D. The distribution of Qa-1b in the skin tissues was detected by immunohistochemistry (400×), MF was the female adult and YF was the female calf. *.P < 0.05, **.P < 0.01, ***.P < 0.001"

Fig. 2

Expression of NKG2A in skins of Mongolia cattle A. Results of the mRNA expression of NKG2A in the skin, Y is calf, M is adult; B. Results of NKG2A protein expression in the skin samples, 1-3 is calf, 4-6 is adult cattle; C. Grayscale value analysis; D. The distribution of NKG2A in the skin tissues was detected by immunohistochemistry(400×), MF is the female adult and YF is the female calf. *.P < 0.05, **.P < 0.01, ***.P < 0.001"

Fig. 3

Qa-1b/NKG2A interaction in skins by immunoprecipitation"

Table 2

Screening of Qa-1b positive monoclonal colonies by ELISA"

Fig. 4

Amino acid sequence analysis of Qa-1b-VHH-A-6 and nanobody P13. Camel source amino acid sequence of VHH; Qa-1. pET28a-Qa-1b-VHH amino acid sequence"

Fig. 5

Plasmids of pET-28a and Qa-1b-VHH-A-6 were cut by BamH Ⅰand EcoR Ⅰ M1 and M2 were 5 000 and 500 bp marker, respectively; 1 channel was the plasmid of pET-28a; Other channels were product of plasmids cut by BamH Ⅰand EcoR Ⅰ"

Fig. 6

Alignment between the sequence of pET28a-Qa-1b-VHH recombinant plasmid and Qa-1b-VHH"

Fig. 7

Protein expression levels in the supernatant after IPTG induction M. Protein molecular mass standard; The expression levels from 1 to 7 were induced by IPTG concentrations of 0, 0.2, 0.4, 0.5, 0.6, 0.8 and 1.0 mmol·L-1, respectively"

Fig. 8

Purification of Qa-1b nanobody In the UV absorption peak, Qa-1b nanobody was eluted at 30% imidazole; A. Bands at 30% imidazole were detected by Western blot; B. The results of affinity chromatography by SDS-PAGE (Coomassie blue staining) showed the single band at 30% imidazole"

Table 3

Detection of binding force between Qa-1b nanobody and Qa-1b"

Fig. 9

The distribution of Qa-1b in canine melanoma tissues was detected by immunohistochemistry (400×)"

Fig. 10

Qa-1b nanobody were used as primary antibodies in Western blotting to detect the expression of Qa-1b in skins samples A. Western blot, 1-3. Calf, 4-6. Adult cattle; B. Grayscale value analysis"

Fig. 11

Effects of Qa-1b nanobody on proliferation and migration of B16 cells A. CCK8 assay was used to detect the inhibitory effect of Qa-1b nanobody on cell proliferation when the concentration of QA-1b nanobody was 120 μg·mL-1; B. Cell scratch assay showed that the addition of Qa-1b nanobody significantly inhibited cell migration; C. The mRNA expressions of MEK1, CDK4, Ras and CyclinD1 were down-regulated by qPCR assay; D, E. Western blot method was used to detect the expression of proliferation-related proteins, and the expression levels of MEK1, CDK4, Ras and Cyclin D1 were reduced, where E was the result of Image J gray analysis. *.P < 0.05, **.P < 0.01, ***.P < 0.001"