ATGGAGCCCGCCGGCCCGGCCCCCGGCCGCCTCGGGCCGCTGCTCTGCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAGGAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTATCGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACGGCTTCTCACCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTGGTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTGGCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGACCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAGTTCTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGGCCTCAACCGTTACAGAGAACAAGGATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCCCACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCCGAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGGTGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAAGAAAGCCCAGGGCTCCACTTCTTCTACAAGGTTGCATGAGCCCGAGAAGAATGCCAGAGAAATAACACAGGACACAAATGATATCACATATGCAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGCGGAGCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCGCAGCCCGCGTCGGAGGACACCCTCACCTATGCTGACCTGGACATGGTCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCAAGCCTGAGCCGTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAGTGA

Table 1. Primers used for cloning of target gene SIRPα
Primers	Sequences
F- SIRPa	ATGGGTCGCGGATCCGAATTCATGGAGCCCGCCGGCCCGGC
R-SIRPa	TCGAGTGCGGCCGCAAGCTTTCAGTGGTGATGGTGATGATGCTTCCTCGGGACCTGGACGCTGGCGTACT

The pET-28a (+) plasmid is a prokaryotic expression vector that contains a His tag at the C-terminal end and a His tag, thrombin cleavage site, and T7 tag at the N-terminal end.² It contains several commonly used enzymatic cleavage sites, which facilitates the cloning of different genes. Unique sites are shown on the circle map in Figure 1 (A). We inserted the SIRPα gene in the MCS region of the pET-28a (+) plasmid as shown in Figure 1 (B).

Figure 1. Pet-28a (+) plasmid mapping. (A) Without SIRPα gene. (B) With SIRPα gene.

MnO₂ nanospheres can effectively catalyze the in situ production of O₂ and relieve hypoxia by reacting with endogenous H₂O₂ to release Mn²⁺. They can also react with GSH in tumor cells to generate GSSH and Mn²⁺. The ability of MnO₂ nanospheres to modulate TME allows them to enhance the antitumor immunotherapy.