Cloning, Expression, and Immunological Characterization of Two Novel Profilins from Artemisia annua

Cloning, Expression, and Immunological Characterization of Two Novel Profilins from Artemisia annua

Allergic diseases have surged globally over recent decades, affecting millions in both developed and developing nations. In China, Artemisia pollen ranks among the most impactful outdoor allergens—according to the first national pollen survey—and is a leading cause of seasonal asthma. Two Artemisia species dominate: Artemisia vulgaris (western China) and Artemisia annua (northern/eastern China, where most people live). While allergens from A. vulgaris (e.g., Art v 1–6) are well-studied, only two from A. annua (Art an 1 and Art an 7) were known until a team of scientists identified two new profilins—small proteins linked to cross-allergies—from A. annua pollen.

The team, led by Qiao-Li Hu, Li-Xiang Zhu, and Zhi-Qiang Xu (co-first authors) from the First Affiliated Hospital of Nanjing Medical University, China Pharmaceutical University, and Nanjing Medical University, used next-generation sequencing and bioinformatics to find the profilins. They amplified the genes via PCR, cloned them into a bacterial vector (pET-28a), and expressed them in Escherichia coli (BL-21). The proteins formed insoluble “inclusion bodies” and were purified using nickel affinity chromatography. The two profilins—profilin 1 and profilin 2—have 399-base-pair genes coding for 133 amino acids each. Their sequences are stored in GenBank (accession numbers MN105099 and MN105100).

Notably, profilin 1’s amino acid sequence matches Art v 4.0101 from A. vulgaris, and profilin 2 matches Art v 4.0202. Both share 65–90% similarity with profilins from other pollens (e.g., ragweed’s Amb a 8, sunflower’s Hel a 2), as shown by evolutionary tree analysis.

To test if these profilins trigger allergic reactions, the team analyzed serum from 200 Artemisia-allergic patients and 16 healthy controls. Using an enzyme-linked immunosorbent assay (ELISA)—a standard test for IgE antibodies—they found 32.5% of patients had IgE reacting to profilin 1 and 27% to profilin 2. These rates align with profilins from other pollens (e.g., birch’s Bet v 2: 22%; sunflower’s Hel a 2: 30.5%), likely due to the proteins’ conserved structure.

Western blotting—another method to detect protein-IgE binding—confirmed results: 7 of 8 positive serum samples bound to profilin 1, and 5 to profilin 2. Some ELISA-positive samples showed no bands, however, because some IgE-binding sites are “conformational” (dependent on 3D structure, which Western blotting can disrupt).

The team also measured how well the profilins blocked IgE binding to crude A. annua extract: profilin 1 inhibited 23.6% of binding, profilin 2 18.6%. Cross-reactivity tests revealed profilin 1 blocked 54.6% of IgE binding to profilin 2 (in one patient’s serum), while profilin 2 blocked 39.4% of binding to profilin 1—meaning the two share IgE-binding sites, a key driver of cross-allergies.

When comparing IgE reactivity to symptoms (asthma, rhinitis, eczema, hay fever, dermatitis, conjunctivitis), the team found no significant differences. Sensitization to these profilins did not link to any specific symptom—important for doctors, as it means the proteins are general markers for Artemisia allergy, not tied to severe cases like asthma alone.

These findings, published in the Chinese Medical Journal in 2021, add critical context to Artemisia allergies in China. The profilins’ similarity to other plant profilins may explain why some people are allergic to multiple pollens (cross-sensitization). For patients, this means more precise allergy testing—using specific components instead of crude extracts—which can improve immunotherapy outcomes. The team notes immunoreactivity to the profilins decreases with age, but more research is needed to confirm this across populations.

All participants gave written informed consent, and the study was approved by the Ethical Committee of the First Affiliated Hospital of Nanjing Medical University (2018-SRFA-094). Funding came from the National Natural Science Foundation of China (grants 81871265, 81771725, U1832212, 81971515).

doi.org/10.1097/CM9.0000000000001309

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