CD28 gene overexpression vector and application thereof

The invention provides a CD28 gene overexpression vector and application thereof. A target biological genome serves as a template, left and right homologous arms of a first intron of an ROSA26 gene, a CD28 gene expression frame and an OCT4 gene promoter are amplified through PCR (polymerase chain reaction), and the left homologous arm, the CD28 gene expression frame, LoxP-locus-containing Cre expression frame and Neo gene expression frame both regulated by the OCT4 gene promoter, the right homologous arm and a negative selection marker DTA are connected sequentially and are constructed to an eukaryotic expression vector to obtain the overexpression vector. The overexpression vector and a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) 9 target vector containing a first intron of a specific target pig ROSA26 gene are transplanted into a fibroblast of a pig fetus, a positive cell serves as a donor cell, an oocyte serves as a recipient cell, and a cloned embryo is obtained according to a somatic cell nuclear transfer technology; the cloned embryo is transplanted into the uterus of a pig for gestation to obtain a transgenic pig with a CD28 gene which is integrated at a fixed point in the first intron of the ROSA26 gene, wherein a marker gene of the transgenic pig is knocked out automatically.